InteractiveFly: GeneBrief

Calnexin 99A: Biological Overview | Developmental Biology | Effects of Mutation | Evolutionary Homologs | References


Gene name - Calnexin 99A

Synonyms -

Cytological map position - 99A7

Function - chaperone

Keywords -eye, Rhodopsin 1 (NinaE), protein folding, calcium ion binding, axon guidance, visual signal transduction

Symbol - Cnx99A

FlyBase ID: FBgn0015622

Genetic map position - 3R

Classification - Calreticulin/calnexin type lectin domain

Cellular location - endoplasmic reticulum transmembrane protein



NCBI links: Precomputed BLAST | EntrezGene
BIOLOGICAL OVERVIEW

In sensory neurons, successful maturation of signaling molecules and regulation of Ca2+ are essential for cell function and survival. A multifunctional role for calnexin has been demonstrated as both a molecular chaperone uniquely required for rhodopsin maturation and a regulator of Ca2+ that enters photoreceptor cells during light stimulation. Mutations in Drosophila calnexin lead to severe defects in rhodopsin (Rh1) expression, whereas other photoreceptor cell proteins are expressed normally. Mutations in calnexin also impair the ability of photoreceptor cells to control cytosolic Ca2+ levels following activation of the light-sensitive TRP channels. Finally, mutations in calnexin lead to retinal degeneration; this degeneration is enhanced by light, suggesting that calnexin's function as a Ca2+ buffer is important for photoreceptor cell survival. These results illustrate a critical role for calnexin in Rh1 maturation and Ca2+ regulation and provide genetic evidence that defects in calnexin lead to retinal degeneration (Rosenbaum, 2006).

G protein-coupled receptors are synthesized on membrane-bound ribosomes and undergo translocation, modification, folding, oligomeric assembly, and quality control in the endoplasmic reticulum (ER). To deal with these complex and error-prone processes, the ER has evolved a system centered around the calnexin family of molecular chaperones; calnexins promotes the proper folding and assembly of newly synthesized glycoproteins. Calnexin is a type I transmembrane protein that, like its soluble ER homolog calreticulin, interacts with the monoglucosylated glycan (Glc1Man7-9GlcNAc2) present on folding intermediates of glycoproteins. Nascent glycoproteins associate with calnexin or calreticulin via a cycle of binding and release (Ellgaard, 2003, Molinar, 2004, Schrag, 2001; Ware, 1995). This cycle is key for ER quality control, since it inhibits aggregation, prevents premature exit from the ER, and exposes glycoproteins to accessory enzymes and folding factors (Ellgaard, 2003). Despite considerable understanding of the calnexin/calreticulin cycle, little is known about the requirement for calnexin in protein processing in vivo (Rosenbaum, 2006).

Rhodopsin is the prototypical member of the large G protein-coupled receptor family. As in vertebrates, Drosophila rhodopsin (Rh1) initiates the phototransduction cascade by interacting with a heterotrimeric G protein, which then activates a distinct effector enzyme, namely phospholipase C (PLC-β). Activation of PLC leads to the opening of the cation-selective TRP and TRPL channels, resulting in a dramatic rise in intracellular Ca2+ (Rosenbaum, 2006 and references therein).

To become functionally active, newly synthesized rhodospin must be precisely folded and successfully navigate the secretory pathway to the phototransducing compartment of the photoreceptor cells, the rhabdomeres. Rhabdomeres consist of numerous tightly packed microvilli containing the phototransduction machinery. The mechanisms that regulate the folding and transport of rhodopsin are essential for photoreceptor cell function and survival, since defects in rhodopsin maturation led to retinal degeneration in both Drosophila and vertebrates. Protein maturation defects characterized in blinding diseases have broad implications, because protein misfolding and aggregation are characteristic of a variety of neurodegenerative diseases, including Alzheimer's disease, Parkinson's disease, and Huntington's disease. This study investigates whether calnexin functions as a chaperone for Rh1 and whether mutations in calnexin lead to neurodegeneration (Rosenbaum, 2006).

In addition to its role as a molecular chaperone, calnexin is thought to bind Ca2+ at two distinct sites. The first resides in the long N-terminal domain localized to the lumen of the ER. The crystal structure of vertebrate calnexin shows that this luminal domain consists of two distinct regions: a compact, globular domain and a proline-rich arm called the P domain. The globular domain is thought to bind a single Ca2+ ion and is also involved in glucose binding (lectin domain) (Schrag, 2001). Although less well defined, several lines of evidence suggest that calnexin may harbor a second Ca2+-binding domain within the highly charged C-terminal cytosolic tail (C domain). Interestingly, this cytosolic domain displays structural similarity to calreticulin's luminal C domain, but is positioned on the opposite, cytosolic side of the ER membrane (Tjoelker, 1994). Calreticulin's C domain displays low-affinity and high-capacity Ca2+ binding and is thought to buffer luminal Ca2+ (Baksh, 1991). While the in vivo role of calnexin's C domain is unknown, these Ca2+-binding properties would make it ideal for buffering high concentrations of cytosolic Ca2+ (Rosenbaum, 2006).

Precise spatial and temporal control over Ca2+ levels is essential for phototransduction in both vertebrates and invertebrates. Furthermore, prolonged elevation of cytosolic Ca2+ can be toxic, leading to cell death. In Drosophila, the light-sensitive TRP and TRPL channels mediate a massive Ca2+ influx into the rhabdomeres that is essential for amplification, rapid-response kinetics, and light adaptation. Ca2+ can rise to about 1 mM in the rhabdomeres and is removed from the rhabdomeres by a combination of the Na+/Ca2+ exchanger (CalX) and diffusion into the cell body, where Ca2+ rises to about 10 μM. The mechanisms controlling Ca2+ in the cell body are poorly understood but presumably include sequestration by the sarco-endoplasmic reticulum Ca2+ ATPase (SERCA) and buffering by cytosolic Ca2+-binding proteins. This study explores the hypothesis that calnexin serves as a buffer for cytosolic Ca2+ following the acute Ca2+ rise during phototransduction and that it may thereby help to prevent Ca2+ toxicity and promote photoreceptor cell survival (Rosenbaum, 2006).

This report demonstrates a multifunctional role for calnexin as both a critical molecular chaperone for Rh1 biosynthesis and a regulator of cytosolic Ca2+. Mutations in calnexin cause retinal degeneration that is enhanced by light, suggesting that calnexin's role in Ca2+ removal may be important for photoreceptor cell survival. These results provide genetic evidence that failure in Rh1 maturation and Ca2+ overload, resulting from defects in calnexin, are responsible for retinal degeneration (Rosenbaum, 2006).

Thus Cnx plays a multifunctional role, serving as a chaperone for Rh1 and a regulator of Ca2+ during phototransduction. Furthermore, genetic evidence shows that failure in Rh1 maturation and Ca2+ overload, resulting from defects in cnx, are responsible for retinal degeneration (Rosenbaum, 2006).

Calnexin serves a key role in glycoprotein folding and quality control in the ER and is thought to interact with a wide variety of newly synthesized proteins. Consistent with calnexin's broad substrate specificity, it is expressed at all stages during development and in the adult. However, cnx mutants are homozygous viable and fertile, indicating that all proteins required for survival and reproduction fold sufficiently in the absence of Cnx. Cnx is, however, essential in the eye. Loss-of-function mutations in cnx lead to defects in Rh1 maturation and cause age-dependent retinal degeneration. Although Cnx is essential for Rh1 biosynthesis, it is not required for the expression or function of other photoreceptor cell proteins. This indicates that even within the eye, Cnx may play a unique role. There are two possible explanations for the specific requirement for Cnx by Rh1: (1) chaperones with redundant functions may compensate for the loss of Cnx; (2) Cnx may serve as a chaperone that is exclusively dedicated to Rh1 (Rosenbaum, 2006).

With the exception of Rh1, photoreceptor cell proteins manage to fold normally in cells lacking calnexin. This could be due to redundancy between folding factors in the ER. For example, calreticulin (located on 3R, 85E) as well as two additional calnexin genes (cnx11 and cnx14) are present in Drosophila. These chaperones may compensate for the lack of calnexin function in all cases except during Rh1 biosynthesis. An alternative conclusion is that Cnx may be exclusively devoted to Rh1. Consistent with this scenario, another chaperone, NinaA, is also expressed in all eight photoreceptor cells yet is specifically dedicated to Rh1 biosynthesis in the R1-6 photoreceptor cells. In addition, a cyclophilin-like protein expressed in the mammalian retina, RanBP2, acts as a specific chaperone for red/green opsin. Given the importance and abundance of rhodopsin, it is plausible that Cnx and NinaA are part of a system of chaperones that is dedicated to its folding and quality control (Rosenbaum, 2006).

Consistent with results presented in this study, calnexin-deficient mice have been generated that are homozygous viable. Half die within the first 2 days, and those that survive are smaller than their littermates, develop severe motor disorders, and display a dramatic loss of large myelinated nerve fibers (Denzel, 2002). Mice contain a single calnexin gene, eliminating the possibility of redundancy between calnexins. However, it is possible that calreticulin is able to partially compensate for the loss of calnexin in utero and in adults, but is unable to compensate for all calnexin functions (Rosenbaum, 2006).

Photoreceptor cells in the cnx mutants display elevated and sustained cytosolic Ca2+ levels following light stimulation, consistent with a role for Cnx in buffering Ca2+. Calnexin's cytosolic domain displays structural similarity to calreticulin's highly charged C domain, but is positioned on the opposite side of the ER membrane (Tjoelker, 1994). Calreticulin's C domain displays low-affinity, high-capacity Ca2+-binding properties and plays a major role in Ca2+ modulation in the lumen of the ER (Baksh, 1991). Given their structural similarity, it is proposed that calnexin's C domain serves a similar function in modulating Ca2+, but in the cyotosol. These low-affinity and high-capacity Ca2+-binding properties would make calnexin's cytosolic domain ideal for buffering high levels of Ca2+ in the photoreceptors. It is proposed that Cnx serves to buffer Ca2+, which diffuses into the cell body after entering via the light-sensitive TRP and TRPL channels (Rosenbaum, 2006).

The Drosophila compound eye has emerged as an important model for unraveling the mechanisms of retinal degeneration and phototransduction. Retinal degeneration can be triggered by mutations in almost every protein that functions in phototransduction. Although the mechanisms for each are not well understood, these mutations can be divided into at least two distinct classes. One involves protein maturation defects, most commonly in rhodopsin, and the other involves a combination of unregulated activities of the phototransduction cascade and/or Ca2+ toxicity. Retinal degeneration caused by defects in rhodopsin folding does not require light activation of phototransduction and is therefore light independent. However, retinal degenerations stemming from unregulated phototransduction or Ca2+ toxicity are dependent on light stimulation of the cascade and opening of the light-sensitive TRP and TRPL channels. Genetic analysis has revealed a number of mutants that fall into one class or the other, but very few that exhibit both properties (Georgiev, 2005). The cnx mutants clearly display defects in Rh1 maturation, and yet they also undergo a light-enhanced retinal degeneration. This indicates that defects in Rh1 maturation are not solely responsible for the retinal degeneration. The results, indicating impaired Ca2+ buffering in the cnx mutants, along with the ability of the norpA mutation to partially prevent degeneration in cnx, suggests that Ca2+ toxicity also contributes to the retinal degeneration. Therefore, it is proposed that the cnx mutant displays characteristics of two distinct classes of retinal degeneration; one involving defects in Rh1 maturation, and the other involving Ca2+ toxicity (Rosenbaum, 2006).

These results provide genetic evidence that failure in Rh1 maturation and Ca2+ overload, resulting from defects in Cnx, are responsible for retinal degeneration. Because calnexin has been localized to the ER of photoreceptor cells in mice, it may play a protein-folding role in the mammalian retina as well as in Drosophila (Frederick, 2001). Furthermore, because Drosophila cnx displays 49% amino acid identity with human calnexin, mutations identified in Drosophila calnexin may be clinically relevant to hereditary human retinal degeneration diseases (Rosenbaum, 2006).


DEVELOPMENTAL BIOLOGY

Embryonic

Calnexin 99A, the sole lineage specific gene in this group, is highly related to its mammalian counterpart, and there are four Drosophila isoforms, coded for by separate genes; only two isoforms have been characterized (Christodoulou, 1997). In the CNS Calnexin 99A expression is restricted to longitudinal and midline glia. Expression is also detected in cells lining the developing gut (Brody, 2002).

Calnexin is thought to play a role in the folding of a large number of proteins. Consistent with this notion, cnx is found by Northern blot to be expressed at all stages during development and in the adult. cnx transcript was detected in wt embryos, larvae, and adult heads and bodies. cnx transcript and Cnx protein were also detected in heads from flies lacking eyes (eya1), indicating that cnx expression is not restricted to the eyes. Despite the widespread expression pattern of cnx, it was not required for viability or fertility of the flies, since the mutants were homozygous viable. Although Rh1 requires Cnx for its expression, Cnx expression is normal in Rh1 mutants (Rosenbaum, 2006).

A targeted gain of function screen in the embryonic CNS of Drosophila involved Cnx99A

EP(3)3522 is located 800 bp upstream from Calnexin 99A (Cnx99A). There are three Calnexin homologs in Drosophila. The first Calnexin identified was mapped to 14D on the X chromosome (also known as CG9906). A second Calnexin-like molecule was mapped to 99A on the third chromosome (also known as CG11958) (Christodoulou, 1997). Upon examination of the Drosophila genome a third Calnexin-like molecule, CG1924, was found at 11A on the X chromosome. The Calnexins encode a calreticulin domain that consists of a low capacity, high affinity calcium binding domain, a globular domain and a high capacity, low affinity calcium binding domain, and a short transmembrane domain (Christodouloul, 1997). Calnexin homologs have been found in a variety of organisms including mammals, plants and yeast. In vertebrates, Calnexin is a membrane protein of the endoplasmic reticulum and is thought to function as a molecular chaperone aiding in the folding of polypeptides (Christodoulou, 1997). Furthermore, mutations in Calnexin have been implicated in a variety of human diseases in which key proteins are not folded properly (Chevet, 1999; McGovern, 2003 and references therein).

Misexpression of Cnx99A generated a fused commissure phenotype as revealed by BP102 immunohistochemistry. The two most medial longitudinal fascicles, as visualized with mAb 1D4, cross and recross the midline leading to a phenotype that is reminiscent of the Robo loss of function phenotype. In order to determine if Robo protein was present, an anti-Robo antibody was used on EP(3)3522/scaGAL4 embryos. Normal levels of Robo protein accumulation were detected: therefore, the inappropriate crossing of the midline by 1D4-positive fascicles is not due to the loss of Robo protein (McGovern, 2003).

To determine if the misexpression phenotype observed was due to changes in cell fate, a set of antibody markers was used to examine neurons and glia. The midline cells appeared wild type at all stages with mAb Sim. Conversely, when midline cells were examined with a riboprobe to NetA, it was found that RNA accumulation in the midline cells began to disappear from some cells at stage 15. NetA RNA became restricted to an even smaller number of midline cells at stage 16, and by stage 17 very few midline cells showed NetA RNA accumulation. This same phenotype was observed with a NetrinB (NetB) riboprobe as well as anti-Wrapper antibody, which identifies the midline glia that ensheathe the commissures. While the over expression of Calnexin99A does not appear to affect early CNS midline determination, it does result in the down-regulation of NetA, NetB and Wrapper at later stages (McGovern, 2003).

A riboprobe to an EST containing Cnx99A revealed strong RNA accumulation throughout the developing CNS at stage 9 in wild-type embryos. At stage 12, accumulation in the gut was observed. Weaker RNA accumulation in the CNS was observed to the end of stage 17. Because there is over 82% nucleotide identity between the three Drosophila Calnexin homologs, including high homology in the 5 and 3 UTRs, it is possible that this riboprobe recognizes all three Calnexin homologs (McGovern, 2003).

The simGAL4 driver was used to investigate the consequence of misexpressing Cnx99A in the CNS midline cells. The BP102 phenotype observed with simGAL4 misexpression was quite similar to the phenotype observed with scaGAL4 misexpression. Misexpression of Cnx99A in all neurons and glia or in CNS midline cells alone resulted in a fuzzy, fused commissure phenotype. Furthermore, NetA, NetB, and Wrapper accumulation were down-regulated with both drivers. Loss of wrapper has previously been shown to result in a fuzzy fused commissure phenotype. In wrapper mutants the midline glial cells develop and migrate properly but at stage 16 they fail to ensheathe the commissural axons and die. In Cnx99A misexpression embryos the BP102 phenotype observed is likely due in part to the down-regulation of Wrapper at later stages. Misexpression of Cnx99A could be affecting CNS midline cell survival directly or indirectly by altering the expression of genes required for CNS midline cell survival such as Wrapper (McGovern, 2003).

Since Cnx99A contains calcium-binding domains the possibility was considered that the inappropriate midline crossing of the 1D4-positive fascicles was due to calcium sequestration in axons. Previous studies have shown that the disruption of Ca2+-calmodulin (CaM) signaling, by sequestering free Ca2+ in the growth cone, results in inappropriate crossing of the midline by the MP1 neuronal pathway. In order to investigate the consequence of misexpressing Cnx99A in midline cells verses neurons the simGAL4 driver was used. If overexpression of Cnx99A in all neurons and midline cells results in reduced calcium levels in the growth cone, it would be expected that restricting expression to just the CNS midline cells would generate a less severe phenotype. This in fact was the case, since only the most medial 1D4 positive fascicle was found to cross the midline when Cnx99A was misexpressed with simGAL4. This is in stark contrast to the severe 1D4 phenotype produced when Cnx99A was misexpressed in all neurons with scaGAL4. The few 1D4-positive fascicles that do cross the midline when Cnx99A was misexpressed with simGAL4 could be a result of reduction in midline guidance cues such as Slit, NetA, and NetB at later stages of CNS development (McGovern, 2003).

Two putative loss of function alleles of Cnx99A were generated. Since Cnx99A is lethal when misexpressed with scaGAL4, EP(3)3522 males were mutagenized and a reversion of the lethality phenotype was sought when scaGAL4 was present. Each putative loss of function allele was placed in trans to a deficiency for the region spanning Cnx99A. No embryonic loss of function phenotypes were observed in the CNS upon examination with mAbs BP102 or 1D4 (McGovern, 2003).


EFFECTS OF MUTATION

By screening the Zuker collection of EMS-mutagenized Drosophila lines, two independent mutants were identified that displayed a severe reduction in Rh1 compared to wild-type (wt). The two mutants were established to be allelic, since the transheterozygotes displayed reduced levels of Rh1. Furthermore, both alleles were shown to be recessive (Rosenbaum, 2006).

To identify the mutant locus responsible for the phenotype, deficiency mapping was used to narrow the cytogenetic location to 99A7 on the third chromosome, corresponding to four genes. This small region was sequenced and mutations were identified in the coding region of calnexin (Christodoulou, 1997) in both alleles. One allele harbored a C to T transition at nucleotide position 544, causing a premature stop codon at glutamine182, and that the other allele harbored a G to A transition at nucleotide position 356, causing a premature stop codon at tryptophan119. Based on cytology, the two alleles were designated cnx99A1 (cnx1) and cnx99A2 (cnx2), respectively. Drosophila calnexin99A (Cnx) displays 49% amino acid identity with human calnexin (chromosome 5q) (Rosenbaum, 2006).

To provide further evidence that mutations in cnx were responsible for the severe reduction in Rh1, the wt cnx gene was introduced into the cnx mutants by using a duplication for 99A that was translocated to the X chromosome [Dp(3;1) B152]; the introduced gene was confirmed to restored the normal function. Consistent with the presence of the stop codons, the cnx mutants displayed severely reduced levels of cnx transcript. In addition, the Cnx protein was absent in both of the cnx mutants, while it was detected in wt flies. Lane 4 confirms that the mutants were allelic. Cnx protein was not detected in the cnx1 and cnx2 mutants when they were crossed to a deficiency (Df) that eliminated 99A (Df(3R)Ptp99A[R3]). These data support the hypothesis that the mutations were in the cnx gene (Rosenbaum, 2006).

Although Rh1 protein levels are severely reduced in the cnx mutants, Rh1 transcript levels are normal in both. These data suggest that Cnx functions post-transcriptionally and are consistent with a role for Cnx as a chaperone in Rh1 biosynthesis (Rosenbaum, 2006).

To investigate the role of Cnx in Rh1 biosynthesis, the kinetics of Rh1 maturation in the cnx1 mutant were assessed. Transgenic flies carrying the Rh1 gene under the control of a heat-shock promoter (hs) were used and tagged with an epitope corresponding to 12 amino acids at the C terminus of bovine rhodopsin (hs-Rh1-bov transgene). The transgene was introduced into wt, cnx1, and ninaAP269 mutant flies. To initiate Rh1 biosynthesis, the transgenic flies were given a 1 hr heat-pulse at 37°C, shifted back to 22°C, and assayed at specific time points. The fate of the heat-induced Rh1 protein was followed by using a monoclonal antibody (1D4) directed to the epitope tag (bov). No Rh1 expression was detected in the flies prior to heat-pulse. In wt flies, Rh1 was initially detected as immature high-MW forms that were converted to the mature low-MW form by 18 hr. In the cnx1 mutant, Rh1 was also initially detected as immature high-MW forms but was significantly reduced by 24 hr. By 48 hr, very little Rh1 was detected, suggesting that most of the Rh1 was degraded (Rosenbaum, 2006).

The failure of Rh1 to mature in the cnx1 mutant was similar to the fate of Rh1 in the ninaA mutant. NinaA is a chaperone specifically required for Rh1 biosynthesis and maturation. As in the cnx1 mutant, Rh1 is initially detected as immature high-MW forms in the ninaA mutant. In contrast to the cnx1 mutant, where most of the Rh1 was degraded, Rh1 accumulates in the immature high-MW form in the ninaA mutant (Rosenbaum, 2006).

The subcellular localization of Rh1 was followed in pulse-chase experiments. In wt flies, by 8 hr following the heat-pulse, Rh1 immunolocalized to the ER in a perinuclear fashion, was detected in a punctate pattern consistent with transport vesicles, and was detected in the rhabdomeres. By 12 hr, more Rh1 was detected in the rhabdomeres, and by 24 hr, mature Rh1 localized solely to the rhabdomeres. This represents the normal progression for Rh1 maturation and transport through the secretory pathway. In the cnx1 mutant, by 8 hr, Rh1 was detected predominantly in the ER. By 12 hr, Rh1 was still most noticeable in the ER. By 24 hr, Rh1 labeling was detected in both the ER and rhabdomeres, but was significantly fainter than wt. These results show that in the cnx mutant, while most Rh1 was degraded, some Rh1 successfully evaded the quality-control mechanisms and was transported to the rhabdomeres. In the ninaA mutants, at 8, 12, and 24 hr, Rh1 was detected primarily in the ER. A very small amount of Rh1 was detected in the rhabdomeres of ninaA mutants, again indicating that a small amount of Rh1 evaded the ER's quality-control system (Rosenbaum, 2006).

It is possible that Cnx and NinaA are part of a protein-processing pathway, ensuring proper folding and quality control of Rh1 during biosynthesis. To gain insights into the epistatic relationship between the two chaperones, mutant flies were created that were defective in both cnx and ninaA. The ninaAP269;cnx1 double mutant displayed severely reduced levels of Rh1, comparable to those seen in the cnx1 mutant alone. These data demonstrate that in the double mutant, Rh1 was effectively degraded rather than accumulating in the ER (as in ninaA). Therefore, cnx is epistatic to ninaA, as the phenotype of the cnx mutation overrides the phenotype of the ninaA mutation in the double mutant (Rosenbaum, 2006).

Because the two chaperones are required for Rh1 biosynthesis, the levels of NinaA protein were examined in the cnx mutants. NinaA levels in cnx were indistinguishable from wt levels, suggesting that the defects in Rh1 were the result of a lack of Cnx, rather than a lack of NinaA (Rosenbaum, 2006).

The association of Cnx with Rh1 was assessed by coimmunoaffinity experiments. Cnx was isolated in a stable complex with Rh1, but did not bind to or elute from the immunoaffinity column in the absence of Rh1. These data indicated that Cnx and Rh1 physically associate in a protein complex, consistent with a role for Cnx as a molecular chaperone for Rh1 (Rosenbaum, 2006).

Examination of the cnx mutants revealed that they displayed an age-related retinal degeneration. Photoreceptor cells in 1-day-old cnx mutants displayed diminished rhabdomere size as compared to wt. They also displayed accumulations of rough ER membranes, dilated Golgi, and various types of deposits. These secretory pathway defects were consistent with a failure in Rh1 maturation. The identity of the deposits is unknown, but they may consist of degraded material within the cells. At 1 month, cnx mutants displayed a dramatic loss of rhabdomeres in the R1-6 photoreceptor cells, while the R7 and R8 photoreceptor cell rhabdomeres remained. The Drosophila compound eye is made up of approximately 750 individual eye units called ommatidia. Each ommatidium contains eight photoreceptor cells. Only R1-6 photoreceptors express Rh1, whereas R7 and R8 cells express a variety of different opsins (Rh3-6). The finding that only R1-6 rhabdomeres degenerated, and not R7 and R8, suggested that the opsins located in R7 and R8 are expressed normally (Rosenbaum, 2006).

To confirm normal expression of the minor rhodopsins in R7 and R8 cells, immunocytochemical analysis of Rh3, Rh4, and Rh5 opsins was performed in the cnx mutants. All three opsins were found to be correctly localized to the rhabdomeres of the R7 and R8 cells, confirming that while Cnx was required by Rh1, it was not required by the R7 and R8 opsins (Rosenbaum, 2006).

Whether ER processing of other photoreceptor proteins was defective in the cnx mutant was examined. Immunocytochemical analysis revealed that another membrane protein, chaoptin, was present at its normal location in the rhabdomeres of the R1-8 photoreceptor cells in both cnx mutants. In addition, immunoblotting analysis was performed to confirm that a number of key phototransduction proteins, including the G protein α subunit (Gqα), the TRP and TRPL channels, PLC-β (norpA), arrestin 1 (Arr1), and arrestin 2 (Arr2) were all expressed at levels indistinguishable from wt. These results demonstrated that while Cnx was required for processing of Rh1, it was not required for expression of these other photoreceptor cell proteins (Rosenbaum, 2006).

To determine the expression pattern for Cnx, polyclonal antibodies were generated that recognized a 97 kDa band in wt flies that was not present in the mutants. Cnx localized to the ER of all eight photoreceptor cells, often to ER cisternae that were tightly associated with the nuclear envelope. The labeling pattern for Cnx was compared to the ER proteins, InsP3R (inositol-1,3,5-trisphosphate receptor) and NinaA. All three proteins were expressed in the ER, but were absent from the rhabdomeres. Although the rhabdomeres of the central R7 photoreceptor cells are labeled by the InsP3R antibody, this labeling is nonspecific. While Cnx protein was uniquely required by Rh1 in the R1-6 cells, it was detected in the ER of all eight photoreceptor cells (Rosenbaum, 2006).

To assess whether the retinal degeneration observed in the cnx mutants is enhanced by light activation of the phototransduction cascade, the cnx mutants were reared for 1 month in constant darkness. These flies displayed a less severe retinal degeneration compared with cnx mutants grown for 1 month on a 12:12 light-dark cycle. Therefore, activation of phototransduction by light enhances the retinal degeneration in the cnx mutants. This result is contrasted to other known mutants defective in Rh1 maturation, such as ninaA, in which the retinal degeneration is light independent. In addition, the ninaA mutants degenerate more slowly compared to the cnx mutants (Rosenbaum, 2006).

The finding that light enhances the retinal degeneration in the cnx mutant led an investigation of whether Ca2+ influx through the light-sensitive channels contributes to the retinal degeneration. Null mutations in the gene encoding the eye-enriched PLC (norpA) eliminate the light-induced Ca2+ influx. norpA;cnx double mutants were generated and it was found that norpA slows down the onset and progression of the retinal degeneration in the cnx mutants. The finding that the retinal degeneration in the cnx mutants is light enhanced and slowed by norpA, in combination with previous findings that calnexin binds Ca2+ (Tjoelker, 1994), prompted a determination of whether Cnx plays a role in modulating Ca2+ in photoreceptor cells (Rosenbaum, 2006).

Current models of phototransduction suggest that virtually all aspects of excitation and adaptation are mediated within the microvilli. Because Cnx is located in the ER, it was predicted that mutations in cnx would not affect the basic light responses, but might cause defects in Ca2+ buffering in the cell body. The basic properties of the light-induced current (LIC) were investigated by using whole-cell patch-clamp recordings of photoreceptors from dissociated ommatidia to record the elementary responses (quantum bumps) representing the response to single-photon absorptions. In addition to wt flies, ninaA mutants were used as controls, because they express low (<1%) levels of functional Rh1 comparable to cnx, but unlike Cnx, the NinaA protein has no predicted Ca2+-binding domains. The most obvious phenotype in both cnx and ninaA was the great reduction in quantum capture (~100- to 200-fold), confirming the reduction in functional rhodopsin. The quantum bump amplitude and waveform in ninaA and cnx were indistinguishable from each other, but both showed a significant (~50%) increase in quantum bump amplitude compared to wt. A similar increase in quantum bump amplitudes has been described in Rh1 hypomorphs. Otherwise, the quantum bump waveform in both cnx and ninaA is indistinguishable from wt. Macroscopic responses to brief test flashes and modest light steps in cnx and ninaA were also indistinguishable from each other, but greatly reduced in sensitivity compared to wt. These results suggested that Cnx does not play a significant role in the basic light response and that, apart from the reduction in Rh1, all other key components of the phototransduction cascade are functional in both cnx and ninaA (Rosenbaum, 2006).

In order to measure Ca2+ levels in the cell body during illumination, the low-affinity Ca2+ indicator dye Fluo-4FF, loaded via the patch pipette, was used. Wild-type, ninaA, and cnx photoreceptor cells were illuminated with the same intensity of 485 nm light, equivalent to ~108 effectively absorbed photons/s in wt flies (though only ~106 photons/s in ninaA and cnx because of the ~100-fold reduction in quantum catch). Even in cnx and ninaA, this corresponds to ~40 effectively absorbed photons/s per microvillus and appeared to be nearly saturating, since a 7.5-fold brighter stimulus only induced slightly larger Ca2+ signals. Fluorescence was measured from the entire cell, where the dominant contribution appears to come from the cell body. After an ~10-20 ms latent period, which allows an estimate of Fmin, the fluorescence increased to a peak after 200-300 ms and then declined to a steady-state plateau. The absolute initial levels reached during this brief latent period in cnx and both wt and ninaA controls were indistinguishable, indicating that there was no systematic difference in dye loading or resting Ca2+ concentrations. However, both the maximum level reached and the plateau in cnx were approximately 2- to 3-fold higher than in either wt or ninaA controls. The comparison with wt is particularly striking, because the effective intensity of illumination was 100-fold times greater in wt flies. A reduction in the sacro-endoplasmic reticulum Ca2+ ATPase (SERCA) or the Na+/Ca2+ exchanger (CalX) was ruled out as being responsible for the increased Ca2+ since both proteins were expressed at wt levels in the cnx mutants. The striking differences in the cytosolic Ca2+ signals between cnx and wt (and ninaA) indicate that Cnx plays an important role in buffering Ca2+ in the cell body (Rosenbaum, 2006).


EVOLUTIONARY HOMOLOGS

Cloning and structure of calnexin

Calnexin is a 90-kDa integral membrane protein of the endoplasmic reticulum (ER). Calnexin binds Ca2+ and may function as a chaperone in the transition of proteins from the ER to the outer cellular membrane. Human calnexin was purified in association with the human interferon-gamma receptor and calnexin cDNA was cloned from placenta. Fragments of calnexin have been prepared as glutathione S-transferase fusion proteins and analyzed for their abilities to bind radioactively labelled Ca2+ and ruthenium red. A subdomain containing four internal repeats binds Ca2+ with the highest affinity. This sequence is highly conserved when compared to calreticulin (a luminal ER protein), an Onchocerca surface antigen, and yeast and plant calnexin homologues. Consequently, this sequence represents a conserved motif for the high-affinity binding of Ca2+, which is clearly distinct from the 'E-F hand' motif. An adjacent subdomain, also highly conserved and containing four internal repeats, fails to bind Ca2+. The carboxyl-terminal, cytosolic domain is highly charged and binds Ca2+ with moderate affinity, presumably by electrostatic interactions. The calnexin amino-terminal domain (residues 1-253) also binds Ca2+, in contrast to the amino-terminal domain of calreticulin, which is relatively less acidic. The cDNA sequences of mouse and rat calnexins have been determined. Comparison of the known mammalian calnexin sequences reveals very high conservation of sequence identity (93%-98%), suggesting that calnexin performs important cellular functions. The gene for human calnexin is located on the distal end of the long arm of human chromosome 5, at 5q35 I (Tjoelker, 1994).

Calnexin as a chaperone

Calnexin is a membrane protein of the endoplasmic reticulum (ER) that functions as a molecular chaperone and as a component of the ER quality control machinery. Calreticulin, a soluble analog of calnexin, is thought to possess similar functions, but these have not been directly demonstrated in vivo. Both proteins contain a lectin site that directs their association with newly synthesized glycoproteins. Although many glycoproteins bind to both calnexin and calreticulin, there are differences in the spectrum of glycoproteins that each binds. Using a Drosophila expression system and the mouse class I histocompatibility molecule as a model glycoprotein, it was found that calreticulin does possess apparent chaperone and quality control functions, enhancing class I folding and subunit assembly, stabilizing subunits, and impeding export of assembly intermediates from the ER. Indeed, the functions of calnexin and calreticulin were largely interchangeable. A soluble form of calnexin (residues 1-387) can functionally replace its membrane-bound counterpart. However, when calnexin was expressed as a soluble protein in L cells, the pattern of associated glycoproteins changed to resemble that of calreticulin. Conversely, membrane-anchored calreticulin binds to a similar set of glycoproteins as calnexin. Therefore, the different topological environments of calnexin and calreticulin are important in determining their distinct substrate specificities (Danilczyk, 2000).

Calnexin and calreticulin are molecular chaperones, which are involved in the protein folding, assembly, and retention/retrieval. Calreticulin-deficiency is lethal in utero, but the contribution of chaperone function to this phenotype is unknown. Protein folding and chaperone function of calnexin was studied in the absence of calreticulin. Protein folding is accelerated and quality control is compromised in calreticulin-deficient cells. Calnexin-substrate association is severely reduced, leading to accumulation of unfolded proteins and a triggering of the unfolded protein response (UPR). PERK and Ire1alpha and eIF2alpha are also activated in calreticulin-deficient cells. The absence of calreticulin can have devastating effects on the function of the others, compromising overall quality control of the secretory pathway and activating UPR-dependent pathways (Knee, 2003).

The endoplasmic (ER) quality control apparatus ensures that misfolded or unassembled proteins are not deployed within the cell, but are retained in the ER and degraded. A glycoprotein-specific system involving the ER lectins calnexin and calreticulin is well documented, but very little is known about mechanisms that may operate for non-glycosylated proteins. A folding mutant of a non-glycosylated membrane protein, proteolipid protein (PLP), was used to examine the quality control of this class of polypeptide. Calnexin associates with newly synthesized PLP molecules, binding stably to misfolded PLP. Calnexin also binds stably to an isolated transmembrane domain of PLP, suggesting that this chaperone is able to monitor the folding and assembly of domains within the ER membrane. Notably, this glycan-independent interaction with calnexin significantly retards the degradation of misfolded PLP. It is proposed that calnexin contributes to the quality control of non-glycosylated polytopic membrane proteins by binding to misfolded or unassembled transmembrane domains, and these findings are discussed in relation to the role of calnexin in the degradation of misfolded proteins (Swanton, 2003).

Terminally misfolded proteins in the endoplasmic reticulum (ER) are retrotranslocated to the cytoplasm and degraded by proteasomes through a mechanism known as ER-associated degradation (ERAD). EDEM, a postulated Man8B-binding protein, accelerates the degradation of misfolded proteins in the ER. Here, EDEM was shown to interact with calnexin, but not with calreticulin, through its transmembrane region. Both binding of substrates to calnexin and their release from calnexin were required for ERAD to occur. Overexpression of EDEM accelerated ERAD by promoting the release of terminally misfolded proteins from calnexin. Thus, EDEM appeared to function in the ERAD pathway by accepting substrates from calnexin (Oda, 2003).

Calreticulin and calnexin are homologous lectins that serve as molecular chaperones for glycoproteins in the endoplasmic reticulum of eukaryotic cells. Calreticulin depletion specifically accelerates the maturation of cellular and viral glycoproteins with a modest decrease in folding efficiency. Calnexin depletion prevents proper maturation of some proteins such as influenza hemagglutinin but does not interfere appreciably with the maturation of several others. A dramatic loss of stringency in the ER quality control with transport at the cell surface of misfolded glycoprotein conformers is only observed when substrate access to both calreticulin and calnexin is prevented. Although not fully interchangeable during assistance of glycoprotein folding, calreticulin and calnexin may work, independently, as efficient and crucial factors for retention in the ER of nonnative polypeptides (Molinari, 2004).

The thiol oxidoreductase endoplasmic reticulum (ER)p57 interacts with newly synthesized glycoproteins through ternary complexes with the chaperones/lectins calnexin or calreticulin. On proteasomal inhibition calnexin and calreticulin concentrate in a pericentriolar endoplasmic reticulum-derived quality control compartment. Surprisingly, ERp57 remains in an endoplasmic reticulum pattern. Using asialoglycoprotein receptors H2a and H2b as models, it was determined in pulse-chase experiments that both glycoproteins initially bind to calnexin and ERp57. However, H2b, which will exit to the Golgi, dissociates from calnexin and remains bound for a longer period to ERp57, whereas the opposite is true for the endoplasmic reticulum-associated degradation substrate H2a that will go to the endoplasmic reticulum-derived quality control compartment. At 15°C, ERp57 colocalizes with H2b adjacent to an endoplasmic reticulum-Golgi intermediate compartment marker. Posttranslational inhibition of glucose excision prolongs association of H2a precursor to calnexin but not to ERp57. Preincubation with a low concentration (15 microg/ml) of the glucosidase inhibitor castanospermine prevents the association of H2a to ERp57 but not to calnexin. This low concentration of castanospermine accelerates the degradation of H2a, suggesting that ERp57, and not calnexin, protects the glycoprotein from degradation. These results suggest an early chaperone-mediated sorting event, with calnexin being involved in the quality control retention of molecules bound for endoplasmic reticulum-associated degradation and ERp57 giving initial protection from degradation and later assisting the maturation of molecules that will exit to the Golgi (Frenkel, 2004).

The UDP-glucose:glycoprotein glucosyltransferase (UGT) is a central player of glycoprotein quality control in the endoplasmic reticulum (ER). UGT reglucosylation of nonnative glycopolypeptides prevents their release from the calnexin cycle and secretion. This study compared the fate of a glycoprotein with a reversible, temperature-dependent folding defect in cells with and without UGT1. Upon persistent misfolding, tsO45 G was slowly released from calnexin and entered a second level of retention-based ER quality control by forming BiP/GRP78-associated disulfide-bonded aggregates. This correlated with loss in the ability to correct misfolding. Deletion of UGT1 did not affect the stringency of ER quality control. Rather, it accelerated release from calnexin and transfer to the second ER quality control level, but it did so after an unexpectedly long lag, showing that cycling in the calnexin chaperone system is not frenetic, as claimed by existing models, and is fully activated only upon persistent glycoprotein misfolding (Molinari, 2005).

Calnexin interaction with oligosaccharides and peptides

Calnexin is a molecular chaperone that resides in the membrane of the endoplasmic reticulum. Most proteins that calnexin binds are N-glycosylated, and treatment of cells with tunicamycin or inhibitors of initial glucose trimming steps interferes with calnexin binding. To test if calnexin is a lectin that binds early oligosaccharide processing intermediates, a recombinant soluble calnexin was created. Incubation of soluble calnexin with a mixture of Glc0-3Man9GlcNAc2 oligosaccharides results in specific binding of the Glc1Man9GlcNAc2 species. Furthermore, Glc1Man5-7GlcNAc2 oligosaccharides bind relatively poorly, suggesting that, in addition to a requirement for the single terminal glucose residue, at least one of the terminal mannose residues is important for binding. To assess the involvement of oligosaccharide-protein interactions in complexes of calnexin and newly synthesized glycoproteins, alpha 1-antitrypsin or the heavy chain of the class I histocompatibility molecule were purified as complexes with calnexin and digested with endoglycosidase H. All oligosaccharides on either glycoprotein were accessible to this probe and could be removed without disrupting the association with calnexin. Furthermore, the addition of 1 M alpha-methyl glucoside or alpha-methyl mannoside had no effect on complex stability. These findings suggest that once complexes between calnexin and glycoproteins are formed, oligosaccharide binding does not contribute significantly to the overall interaction. However, it is likely that the binding of Glc1Man9GlcNAc2 oligosaccharides is a crucial event during the initial recognition of newly synthesized glycoproteins by calnexin (Ware, 1995).

Calnexin and calreticulin are homologous molecular chaperones of the endoplasmic reticulum. Their binding to newly synthesized glycoproteins is mediated, at least in part, by a lectin site that recognizes the early N-linked oligosaccharide processing intermediate, Glc1Man9GlcNAc2. This study compared the oligosaccharide binding specificities of calnexin and calreticulin in an effort to determine the basis for reported differences in their association with various glycoproteins. Using mono-, di-, and oligosaccharides to inhibit the binding of Glc1Man9GlcNAc2 to calreticulin and to a truncated, soluble form of calnexin, it was shown that the entire Glc alpha 1-3Man alpha 1-2Man alpha 1-2Man structure, extending from the alpha 1-3 branch point of the oligosaccharide core, is recognized by both proteins. Furthermore, analysis of the binding of monoglucosylated oligosaccharides containing progressively fewer mannose residues suggests that for both proteins the alpha 1-6 mannose branch point of the oligosaccharide core is also essential for recognition. Consistent with their essentially identical substrate specificities, calnexin and calreticulin exhibit the same relative affinities when competing for binding to the Glc1Man9GlcNAc2 oligosaccharide. Thus, differential glycoprotein binding cannot be attributed to differences in the lectin specificities or binding affinities of calnexin and calreticulin. The effects of ATP, calcium, and disulfide reduction on the lectin properties of calnexin and calreticulin was examined. Whereas oligosaccharide binding is only slightly enhanced for both proteins in the presence of high concentrations of a number of adenosine nucleotides, removal of bound calcium abrogates oligosaccharide binding, an effect that is largely reversible upon readdition of calcium. Disulfide reduction had no effect on oligosaccharide binding by calnexin, but binding by calreticulin was inhibited by 70%. Finally, deletion mutagenesis of calnexin and calreticulin identified a central proline-rich region characterized by two tandem repeat motifs as a segment capable of binding oligosaccharide. This segment bears no sequence homology to the carbohydrate recognition domains of other lectins (Vassilakos, 1998).

The three-dimensional structure of the lumenal domain of the lectin-like chaperone calnexin determined to 2.9Å resolution reveals an extended 140Å arm inserted into a beta sandwich structure characteristic of legume lectins. The arm is composed of tandem repeats of two proline-rich sequence motifs which interact with one another in a head-to-tail fashion. Identification of the ligand binding site establishes calnexin as a monovalent lectin, providing insight into the mechanism by which the calnexin family of chaperones interacts with monoglucosylated glycoproteins (Schrag, 2001).

Calnexin and calreticulin are molecular chaperones of the endoplasmic reticulum that bind to newly synthesized glycoproteins in part through a lectin site specific for monoglucosylated [Glc(1)Man(7-9)GlcNAc(2)] oligosaccharides. In addition to this lectin-oligosaccharide interaction, in vitro studies have demonstrated that calnexin and calreticulin can bind to polypeptide segments of both glycosylated and nonglycosylated proteins. However, the in vivo relevance of this latter interaction has been questioned. This study examined whether polypeptide-based interactions occur between calnexin and its substrates in vivo using the glucosidase inhibitor castanospermine or glucosidase-deficient cells to prevent the formation of monoglucosylated oligosaccharides. If care is taken to preserve weak interactions, the block in lectin-oligosaccharide binding leads to the loss of some calnexin-substrate complexes, but many others remain readily detectable. Furthermore, calnexin is capable of associating in vivo with a substrate that completely lacks Asn-linked oligosaccharides. The binding of calnexin to proteins that lack monoglucosylated oligosaccharides could not be attributed to nonspecific adsorption nor to its inclusion in protein aggregates. It is concluded that both lectin-oligosaccharide and polypeptide-based interactions occur between calnexin and diverse proteins in vivo and that the strength of the latter interaction varies substantially between protein substrates (Danilczyk, 2001).

Calnexin and calreticulin are membrane-bound and soluble chaperones, respectively, of the endoplasmic reticulum (ER) which interact transiently with a broad spectrum of newly synthesized glycoproteins. In addition to sharing substantial sequence identity, both calnexin and calreticulin bind to monoglucosylated oligosaccharides of the form Glc(1)Man(5-9)GlcNAc(2), interact with the thiol oxidoreductase, ERp57, and are capable of acting as chaperones in vitro to suppress the aggregation of non-native proteins. To understand how these diverse functions are coordinated, the lectin, ERp57 binding, and polypeptide binding sites of calnexin and calreticulin were localized. Recent structural studies suggest that both proteins consist of a globular domain and an extended arm domain comprised of two sequence motifs repeated in tandem. The results indicate that the primary lectin site of calnexin and calreticulin resides within the globular domain, but the results also point to a much weaker secondary site within the arm domain that lacks specificity for monoglucosylated oligosaccharides. For both proteins, a site of interaction with ERp57 is centered on the arm domain, which retains approximately 50% of binding compared with full-length controls. This site is in addition to a Zn(2+)-dependent site located within the globular domain of both proteins. Finally, calnexin and calreticulin suppress the aggregation of unfolded proteins via a polypeptide binding site located within their globular domains but require the arm domain for full chaperone function. These findings are integrated into a model that describes the interaction of glycoprotein folding intermediates with calnexin and calreticulin (Leach, 2002).

Calnexin is an endoplasmic reticulum chaperone that binds to substrates containing monoglucosylated oligosaccharides. Whether calnexin can also directly recognize polypeptide components of substrates is controversial. Calnexin displays significant conformational lability for a chaperone and heat treatment and calcium depletion induces the formation of calnexin dimers and higher order oligomers. These conditions enhance the chaperone activity of calnexin toward glycosylated and non-glycosylated major histocompatibility complex (MHC) class I heavy chains, and enhance calnexin binding to MHC class I heavy chains. In contrast to these observations, calnexin binding to oligosaccharide substrates has been reported to be impaired under calcium-depleting conditions. Calnexin dimers are induced in HeLa cells upon heat shock and under calcium-depleting conditions, and heat shock enhances calnexin binding to MHC class I heavy chains in HeLa cells. Virus-induced endoplasmic reticulum stress also results in the appearance of calnexin dimers. Tunicamycin treatment of HeLa cells induces a slow accumulation of calnexin dimers, the appearance of which correlates with enhanced calnexin binding to deglycosylated MHC class I heavy chains. In vitro, the presence of calnexin-specific oligosaccharides inhibits the formation of calnexin dimers and higher order structures. Together, these data indicate that polypeptide binding is favored by conditions that induce partial unfolding of calnexin monomers, whereas oligosaccharide binding is favored by conditions that enhance the structural stability (folding) of calnexin monomers. Conditions that induce the calnexin 'polypeptide-binding' conformation also induce self-association of calnexin if the concentration is sufficiently high; however, calnexin dimerization/oligomerization per se is not essential for polypeptide substrate binding (Thammavongsa, 2005).

Calnexin in yeast

In mammalian cells, the calnexin/calreticulin chaperones play a key role in glycoprotein folding and its control within the endoplasmic reticulum (ER), by interacting with folding intermediates via their monoglucosylated glycans. This lectin activity has been mapped in mammalian calnexin/calreticulin chaperones to the central region, which is a highly conserved feature of calnexin/calreticulin molecules across species. The central domain has also been implicated in Ca(2+) binding, and it has been proposed to be involved in the regulation of calcium homeostasis in the ER. This study shows that although the Schizosaccharomyces pombe calnexin is essential for viability, cells lacking its 317-amino-acid highly conserved central region are viable under normal growth conditions. However, the central region appears to be necessary for optimal growth under high ER-stress, suggesting that this region is important under extreme folding situations (such as DTT and temperature). The minimal length of calnexin required for viability spans the C-terminal 123 residues. Furthermore, cells with the central domain of the protein deleted were affected in their morphology at 37° C, probably due to a defect in cell wall synthesis, although these mutant cells exhibited the same calcium tolerance as wild-type cells at 30° C (Elagoz, 1999).

Calnexin is a molecular chaperone playing key roles in protein folding and the quality control of this process in the endoplasmic reticulum. cnx1+, the gene encoding the calnexin homologue in Schizosaccharomyces pombe, is essential for viability. A particular cnx1 mutant induces a novel mechanism allowing the survival of S. pombe cells in the absence of calnexin/Cnx1p. Calnexin independence is dominant in diploid cells and is inherited in a non-Mendelian manner. Remarkably, this survival pathway, bypassing the necessity for calnexin, can be transmitted by transformation of cell extracts into a wild-type naive strain, thus implicating a non-chromosomal factor. Nuclease and UV treatments of cells extracts did not obliterate transmission of calnexin independence by transformation. However, protease digestion of extracts did reduce the appearance of calnexin-independent cells, indicating that a protein element is required for calnexin-less viability. A model is discussed in which this calnexin-less survival mechanism would be activated and perpetuated by a protein component acting as a genetic element (Collin, 2004).

Calnexin function in C. elegans

Calnexin, a type I integral Ca(2+)-binding protein in the endoplasmic reticulum (ER) membrane, has been implicated in various biological functions including chaperone activity, calcium homeostasis, phagocytosis, and ER stress-induced apoptosis. Caenorhabditis elegans CNX-1 is expressed in the H-shaped excretory cell, intestine, dorsal and ventral nerve cord, spermatheca, and head and tail neurons throughout development. A cnx-1 null mutant displays temperature-sensitive developmental and reproductive defects, and retarded growth under stress. Moreover, a double knockout mutant of calnexin and calreticulin exhibits additive severe defects. Interestingly, both cnx-1 transcript and protein levels are elevated under stress conditions suggesting that CNX-1 may be important for stress-induced chaperoning functions in C. elegans. Glycosidase treatment and site-directed mutagenesis confirm that CeCNX-1 is N-glycosylated at two asparagine residues of Asn(203) and Asn(571). When transgenic animals from cnx-1 mutant were generated, a glycosylation defective construct failed to rescue phenotypes of cnx-1 mutant suggesting that glycosylation is important for calnexin's functions in C. elegans (Lee, 2005).

Proper folding and maintenance of the native structure are central to protein function and are assisted by a family of proteins called chaperones. Calreticulin and calnexin are ER resident chaperones well conserved from worm to human. Calreticulin/calnexin knock-out mice exhibit a severe phenotype, whereas in Caenorhabditis elegans, calreticulin (crt-1jh101)- and calnexin (cnx-1nr2009)-null mutant worms exhibit only a mild phenotype, suggesting the possible existence of alternative chaperone machinery that can compensate for the deficiency of calreticulin and/or calnexin. In order to rapidly identify the compensatory chaperone components involved in this process, the proteome of crt-1jh101 mutants and crt-1jh101;cnx-1nr2009 double mutants was examined. When grown at 20° C, it was found that five proteins were up-regulated and two proteins were down-regulated in crt-1jh101 mutants; nine proteins were up-regulated and five proteins were down-regulated in crt-1jh101;cnx-1nr2009 double mutants. In addition, elevation of the cultivation temperature to 25° C, which is still permissive to growth but causes specific defects in mutants, led to the identification of several additional proteins. Interestingly, the consistent increment of heat shock protein-70 family members (hsp70) together with protein disulfide isomerase (PDI) at all the examined conditions suggests the possible compensatory function imparted by hsp70 and PDI family members in the absence of calreticulin and/or calnexin (Lee, 2006).

Mutation of mammalian calnexin

Calnexin is a ubiquitously expressed type I membrane protein that is exclusively localized in the endoplasmic reticulum (ER). In mammalian cells, calnexin functions as a chaperone molecule and plays a key role in glycoprotein folding and quality control within the ER by interacting with folding intermediates via their monoglucosylated glycans. In order to gain more insight into the physiological roles of calnexin, calnexin gene-deficient mice were generated. Despite its profound involvement in protein folding, calnexin is not essential for mammalian-cell viability in vivo: calnexin gene knockout mice were carried to full term, although 50% died within 48 h and the majority of the remaining mice had to be sacrificed within 4 weeks, with only a very few mice surviving to 3 months. Calnexin gene-deficient mice were smaller than their littermates and showed very obvious motor disorders, associated with a dramatic loss of large myelinated nerve fibers. Thus, the critical contribution of calnexin to mammalian physiology is tissue specific (Danzel, 2002).

Calnexin and channels

To identify factors involved in the expression of ligand-gated ion channels, nicotinic acetylcholine receptors were expressed in HEK cells to characterize roles for oligosaccharide trimming, calnexin association, and targeting to the proteasome. The homologous subunits of the acetylcholine receptor traverse the membrane four times, contain at least one oligosaccharide, and are retained in the endoplasmic reticulum until completely assembled into the circular arrangement of subunits of delta-alpha-gamma-alpha-beta to enclose the ion channel. Calnexin is associated with unassembled subunits of the receptor, but appears to dissociate when subunits are assembled in various combinations. The glucosidase inhibitor castanospermine was used to block oligosaccharide processing, and thereby inhibit calnexin's interaction with the oligosaccharides in the receptor subunits. Castanospermine treatment reduces the association of calnexin with the alpha-subunit of the receptor, and diminishes the intracellular accumulation of unassembled receptor subunit protein. However, treatment with castanospermine does not appear to alter subunit folding or assembly. In contrast, co-treatment with proteasome inhibitors and castanospermine enhances the accumulation of polyubiquitin-conjugated alpha-subunits, and generally reverses the castanospermine induced loss of alpha-subunit protein. Co-transfection of cDNAs encoding the alpha- and delta-subunits, which leads to the expression of assembled alpha- and delta- subunits, also inhibits the loss of alpha-subunits expressed in the presence of castanospermine. Taken together, these observations indicate that calnexin association reduces the degradation of unassembled receptor subunits in the ubiquitin-proteasome pathway (Keller, 1998).

Calnexin is part of an ER chaperone system that monitors and promotes the proper folding and assembly of glycosylated membrane proteins. To investigate the role of calnexin in the biogenesis of the voltage-dependent Shaker K+ channel, wild-type and mutant Shaker proteins were expressed in mammalian cells. Association with calnexin was assayed by coimmunoprecipitation. Calnexin interacts transiently with wild-type Shaker protein in the ER. In contrast, calnexin failed to associate with an unglycosylated Shaker mutant that makes active, cell surface channels. Therefore, glycosylation of Shaker protein is required for association with calnexin, but calnexin is not required for the proper folding and assembly of Shaker channels. Whether calnexin is involved in the ER retention of mutant Shaker proteins defective in subunit folding, assembly, or pore formation, was investigated. Each of the mutant proteins associated transiently with calnexin during biogenesis. Calnexin dissociated from wild-type and mutant proteins with similar time courses. Thus, non-native Shaker proteins escape the folding sensor of the calnexin chaperone system. Furthermore, stable association with calnexin is not the mechanism by which these mutant proteins are retained in the ER. These results indicate that calnexin is not involved in the quality control of subunit folding, assembly, or pore formation in Shaker K+ channels (Nagaya, 1999).

An unglycosylated form of the Shaker potassium channel protein is retained in the endoplasmic reticulum (ER) and degraded by proteasomes in mammalian cells despite apparently normal folding and assembly. These results suggest that channel proteins with a native structure can be substrates for ER-associated degradation. This hypothesis was tested using the wild-type Shaker protein. Wild-type Shaker is degraded by cytoplasmic proteasomes when it is trapped in the ER and prevented from interacting with calnexin. Neither condition alone is sufficient to destabilize the protein. Proteasomal degradation of the wild-type protein is abolished when ER mannosidase I trimming of the core glycan is inhibited. These results indicate that transient interaction with calnexin provides long-term protection from ER-associated degradation (Khanna, 2004).

Long QT syndrome type 2 is caused by mutations in the human ether-a-go-go-related gene (hERG). The N470D mutation is retained in the endoplasmic reticulum (ER) but can be rescued to the plasma membrane by hERG channel blocker E-4031. The mechanisms of ER retention and how E-4031 rescues the N470D mutant are poorly understood. In this study, the interaction of hERG channels with the ER chaperone protein calnexin was investigated. Using coimmunoprecipitation, it was shown that the immature forms of both wild type hERG and N470D associate with calnexin. The association requires N-linked glycosylation of hERG channels. Pulse-chase analysis revealed that N470D has a prolonged association with calnexin compared with wild type hERG and that E-4031 shortens the time course of calnexin association with N470D. To test whether the prolonged association of N470D with calnexin is due to defective folding of mutant channels, hERG channel folding was studied using the trypsin digestion method. It was found that N470D and the immature form of wild type hERG are more sensitive to trypsin digestion than the mature form of wild type hERG. In the presence of E-4031, N470D becomes more resistant to trypsin even when its ER-to-Golgi transport is blocked by brefeldin A. These results suggest that defective folding of N470D contributes to its prolonged association with calnexin and ER retention and that E-4031 may restore proper folding of the N470D channel leading to its cell surface expression (Gong, 2005).

Calnexin and major histocompatibility complex molecules

A mammalian semipermeabilized cell system has been establishes that faithfully reconstitutes the proteasome-mediated degradation of major histocompatibility complex Class I heavy chain. Degradation was shown to require unfolding of the protein and was shown to be cytosol- and ATP-dependent and dislocation and degradation was shown to require proteasome activity. When the interaction of heavy chain with calnexin is prevented, the rate of degradation is accelerated, suggesting that an interaction with calnexin stabilizes the heavy chain. Stabilization of the heavy chain to degradation was also achieved either by preventing mannose trimming or by removal of the N-linked glycosylation site. This demonstrates that glycosylation and mannose trimming are required to ensure heavy chain degradation. When degradation or mannose trimming is inhibited, the heavy chain forms a prolonged interaction with immunoglobulin heavy chain binding protein, ERp57, and protein disulfide isomerase. Taken together, these results indicate that calnexin association and mannose trimming provide a mechanism to regulate the folding, assembly, and degradation of glycoproteins entering the secretory pathway (Wilson, 2000).

Members of the CD1 family of membrane glycoproteins can present antigenic lipids to T lymphocytes. Like major histocompatibility complex class I molecules, they form a heterodimeric complex of a heavy chain and beta(2)-microglobulin [beta(2)m] in the endoplasmic reticulum (ER). Binding of lipid antigens, however, takes place in endosomal compartments, similar to class II molecules, and on the plasma membrane. Unlike major histocompatibility complex class I or CD1b molecules, which need beta(2)m to exit the ER, CD1d can be expressed on the cell surface as either a free heavy chain or associated with beta(2)m. These differences led to an investigation of early events of CD1d biosynthesis and maturation and the role of ER chaperones in its assembly. CD1d associates in the ER with both calnexin and calreticulin and with the thiol oxidoreductase ERp57 in a manner dependent on glucose trimming of its N-linked glycans. Complete disulfide bond formation in the CD1d heavy chain was substantially impaired if the chaperone interactions were blocked by the glucosidase inhibitors castanospermine or N-butyldeoxynojirimycin. The formation of at least one of the disulfide bonds in the CD1d heavy chain is coupled to its glucose trimming-dependent association with ERp57, calnexin, and calreticulin (Kang, 2002).

Calnexin is a membrane-bound lectin of the endoplasmic reticulum (ER) that binds transiently to newly synthesized glycoproteins. By interacting with oligosaccharides of the form Glc(1)Man(9)GlcNAc(2), calnexin enhances the folding of glycoprotein substrates, retains misfolded variants in the ER, and in some cases participates in their degradation. Calnexin has also been shown to bind polypeptides in vivo that do not possess a glycan of this form and to function in vitro as a molecular chaperone for nonglycosylated proteins. To test the relative importance of the lectin site compared with the polypeptide-binding site, six calnexin mutants defective in oligosaccharide binding were generated using site-directed mutagenesis. Expressed as glutathione S-transferase fusions, these mutants were still capable of binding ERp57, a thiol oxidoreductase, and preventing the aggregation of a nonglycosylated substrate, citrate synthase. They were, however, unable to bind Glc(1) Man(9)GlcNAc(2) oligosaccharide and were compromised in preventing the aggregation of the monoglucosylated substrate jack bean alpha-mannosidase. Two of these mutants were then engineered into full-length calnexin for heterologous expression in Drosophila cells along with the murine class I histocompatibility molecules K(b) and D(b) as model glycoproteins. In this system, lectin site-defective calnexin was able to replace wild type calnexin in forming a complex with K(b) and D(b) heavy chains and preventing their degradation. Thus, at least for class I molecules, the lectin site of calnexin is dispensable for some of its chaperone functions (Leach, 2004).

This study investigated how asparagine (N)-linked glycosylation affects assembly of acetylcholine receptors (AChRs) in the endoplasmic reticulum (ER). Block of N-linked glycosylation inhibits AChR assembly whereas block of glucose trimming partially blocks assembly at the late stages. Removal of each of seven glycans has a distinct effect on AChR assembly, ranging from no effect to total loss of assembly. Because the chaperone calnexin associates with N-linked glycans, calnexin interactions with AChR subunits was examined. Calnexin rapidly associates with 50% or more of newly synthesized AChR subunits, but not with subunits after maturation. Block of N-linked glycosylation or trimming does not alter calnexin-AChR subunit associations nor do subunit mutations prevent N-linked glycosylation. Additionally, calnexin associations with subunits lacking N-linked glycans occurs without subunit aggregation or misfolding. These data indicate that calnexin associates with AChR subunits without N-linked glycan interactions. Furthermore, calnexin-subunit associations only occur early in AChR assembly and have no role in events later that require N-linked glycosylation (Wanamaker, 2005).

Calnexin and Apolipoprotein(a)

Apolipoprotein(a) [apo(a)] is a component of atherogenic lipoprotein(a) [Lp(a)]. Differences in the extent of endoplasmic reticulum (ER) associated degradation (ERAD) of apo(a) allelic variants contribute to the >1000-fold variation in plasma Lp(a) levels. Using human apo(a) transgenic mouse hepatocytes, the role of the ER chaperones calnexin (CNX) and calreticulin (CRT), and ER mannosidase I in apo(a) intracellular targeting, was analyzed. Co-immunoprecipitation and pulse-chase analyses revealed similar kinetics of apo(a) interaction with CNX and CRT, peaking 15-30 min after apo(a) synthesis. Trapping of apo(a) N-linked glycans in their monoglucosylated form, by posttranslational inhibition of ER glucosidase activity with castanospermine (CST), enhances apo(a)-CNX/CRT interaction and prevents both apo(a) secretion and ERAD. Delay of CST addition until 20 or 30 min after apo(a) synthesis [when no apo(a) had yet undergone degradation or Golgi-specific carbohydrate modification] allows a portion of apo(a) to be secreted or degraded. These results are consistent with a transient apo(a)-CNX/CRT association and suggest that events downstream of CNX/CRT interaction determine apo(a) intracellular targeting. Inhibition of ER mannosidase I with deoxymannojirimycin or kifunensine has no effect on apo(a) secretion, but inhibits proteasome-mediated apo(a) ERAD even under conditions where apo(a)-CNX/CRT interaction is prevented. These results suggest a role for an additional, mannose-specific, ER lectin in targeting secretory proteins to the proteasome for destruction (Wang, 2000).

Calnexin and viruses

Calnexin and calreticulin are homologous lectin chaperones that assist maturation of cellular and viral glycoproteins in the mammalian endoplasmic reticulum. Calnexin and calreticulin share the same specificity for monoglucosylated protein-bound N-glycans but associate with a distinct set of newly synthesized polypeptides. Most calnexin substrates do not associate with calreticulin even upon selective calnexin inactivation, while BiP associates more abundantly with nascent polypeptides under these conditions. Calreticulin associates more abundantly with orphan calnexin substrates only in infected cells and preferentially with polypeptides of viral origin, showing stronger dependence of model viral glycoproteins on endoplasmic reticulum lectins. This may explain why inactivation of the calnexin cycle affects viral replication and infectivity but not viability of mammalian cells (Pieren, 2005).


REFERENCES

Search PubMed for articles about Drosophila Calnexin 99A

Baksh, S. and Michalak, M. (1991). Expression of calreticulin in Escherichia coli and identification of its Ca2+ binding domains. J. Biol. Chem. 266(32): 21458-65. 1939178

Brody, T., Stivers, C., Nagle, J. and Odenwald, W. F. (2002). Identification of novel Drosophila neural precursor genes using a differential embryonic head cDNA screen. Mech. Dev. 113(1): 41-59. 11900973

Chevet, E., Jakob, C. A., Thomas, D. Y. and Bergeron, J. J. (1999). Calnexin family members as modulators of genetic diseases. Semin. Cell Dev. Biol. 10(5): 473-80. 10597630

Christodoulou, S., et al. (1997). Nucleotide sequence of a Drosophila melanogaster cDNA encoding a calnexin homologue. Gene 191(2): 143-8. 9218712

Collin, P., et al. (2004). A non-chromosomal factor allows viability of Schizosaccharomyces pombe lacking the essential chaperone calnexin. J. Cell Sci. 117(Pt 6): 907-18. 14963023

Danilczyk, U. G., Cohen-Doyle, M. F. and Williams, D. B. (2000). Functional relationship between calreticulin, calnexin, and the endoplasmic reticulum luminal domain of calnexin. J. Biol. Chem. 275(17): 13089-97. 10777614

Danilczyk, U. G. and Williams, D. B. (2001). The lectin chaperone calnexin utilizes polypeptide-based interactions to associate with many of its substrates in vivo. J. Biol. Chem. 276(27): 25532-40. 11337494

Denzel A, Molinari M, Trigueros C, Martin JE, Velmurgan S, Brown S, Stamp G, Owen MJ. (2002). Early postnatal death and motor disorders in mice congenitally deficient in calnexin expression. Mol. Cell. Biol. 22(21): 7398-404. 12370287

Elagoz, A., Callejo, M., Armstrong, J. and Rokeach, L. A. (1999). Although calnexin is essential in S. pombe, its highly conserved central domain is dispensable for viability. J. Cell Sci. 112: 4449-60. 10564662

Ellgaard, L. and Helenius, A. (2003) Quality control in the endoplasmic reticulum. Nature Rev. Mol. Cell Biol. 4: 181-191. 12612637

Frederick, J. M., et al. (2001). Mutant rhodopsin transgene expression on a null background. Invest. Ophthalmol. Vis. Sci. 42(3): 826-33. 11222546

Frenkel, Z., Shenkman, M., Kondratyev, M. and Lederkremer, G. Z. (2004). Separate roles and different routing of calnexin and ERp57 in endoplasmic reticulum quality control revealed by interactions with asialoglycoprotein receptor chains. Mol. Biol. Cell 15(5): 2133-42. 14978212

Georgiev, P., Garcia-Murillas, I., Ulahannan, D., Hardie, R. C. and Raghu, P. (2005). Functional INAD complexes are required to mediate degeneration in photoreceptors of the Drosophila rdgA mutant. J. Cell Sci. 118(Pt 7): 1373-84. 15755798

Gong, Q., Jones, M. A. and Zhou, Z. (2005). Mechanisms of pharmacological rescue of trafficking-defective hERG mutant channels in human long QT syndrome. J. Biol. Chem. 281(7): 4069-74. 16361248

Kang, S. J. and Cresswell, P. (2002). Calnexin, calreticulin, and ERp57 cooperate in disulfide bond formation in human CD1d heavy chain. J. Biol. Chem. 277(47): 44838-44. 12239218

Keller, S. H., Lindstrom, J. and Taylor, P. (1998). Inhibition of glucose trimming with castanospermine reduces calnexin association and promotes proteasome degradation of the alpha-subunit of the nicotinic acetylcholine receptor. J. Biol. Chem. 273: 17064-17072. 9642271

Khanna, R., Lee, E. J. and Papazian, D. M. (2004). Transient calnexin interaction confers long-term stability on folded K+ channel protein in the ER. J. Cell Sci. 117: 2897-908. 15161937

Knee, R., et al. (2003). Compromised calnexin function in calreticulin-deficient cells. Biochem. Biophys. Res. Commun. 304(4): 661-6. 12727205

Leach, M. R., Cohen-Doyle, M. F., Thomas, D. Y. and Williams, D. B. (2002). Localization of the lectin, ERp57 binding, and polypeptide binding sites of calnexin and calreticulin. J. Biol. Chem. 277(33): 29686-97. 12052826

Leach, M. R. and Williams, D. B. (2004). Lectin-deficient calnexin is capable of binding class I histocompatibility molecules in vivo and preventing their degradation. J. Biol. Chem. 279(10): 9072-9. 14699098

Lee, W., et al. (2005). Caenorhabditis elegans calnexin is N-glycosylated and required for stress response. Biochem. Biophys. Res. Commun. 338(2): 1018-30. 16256074

Lee, W., et al. (2006). Alternative chaperone machinery may compensate for calreticulin/calnexin deficiency in Caenorhabditis elegans. Proteomics 6(4): 1329-39. 16404716

McGovern, V. L., Pacak, C. A., Sewell, S. T., Turski, M. L. and Seeger, M. A. (2003). A targeted gain of function screen in the embryonic CNS of Drosophila. Mech. Dev. 120(10): 1193-207. 14568107

Molinari, M., et al. (2004). Contrasting functions of calreticulin and calnexin in glycoprotein folding and ER quality control. Mol. Cell 13(1): 125-35. 14731400

Molinari, M., Galli, C., Vanoni, O., Arnold, S. M. and Kaufman, R. J. (2005). Persistent glycoprotein misfolding activates the glucosidase II/UGT1-driven calnexin cycle to delay aggregation and loss of folding competence. Mol. Cell 20(4): 503-12. 16307915

Nagaya, N., Schulteis, C. T. and Papazian, D. M. (1999). Calnexin associates with Shaker K+ channel protein but is not involved in quality control of subunit folding or assembly. Recept. Channels 6: 229-239. 10412717

Oda, Y., Hosokawa, N., Wada, I. and Nagata, K. (2003). EDEM as an acceptor of terminally misfolded glycoproteins released from calnexin. Science 299(5611): 1394-7. 12610305

Pieren, M., Galli, C., Denzel, A. and Molinari M. (2005). The use of calnexin and calreticulin by cellular and viral glycoproteins. J. Biol. Chem. 280(31): 28265-71. 15951445

Rosenbaum, E. E., Hardie, R. C. and Colley, N. J. (2006). Calnexin is essential for rhodopsin maturation, Ca2+ regulation, and photoreceptor cell survival. Neuron 49(2): 229-41. 16423697

Schrag, J. D., et al. (2001). The structure of calnexin, an ER chaperone involved in quality control of protein folding. Mol. Cell 8: 633-644. 11583625

Swanton, E., High, S. and Woodman, P. (2003). Role of calnexin in the glycan-independent quality control of proteolipid protein. EMBO J. 22(12): 2948-58. 12805210

Thammavongsa, V., Mancino, L. and Raghavan, M. (2005). Polypeptide substrate recognition by calnexin requires specific conformations of the calnexin protein. J. Biol. Chem. 280(39): 33497-505. 16061483

Tjoelker, L. W., et al. (1994). Human, mouse, and rat calnexin cDNA cloning: identification of potential calcium binding motifs and gene localization to human chromosome 5. Biochemistry 33: 3229-3236. 8136357

Vassilakos, A., Michalak, M., Lehrman, M. A. and Williams, D. B. (1998). Oligosaccharide binding characteristics of the molecular chaperones calnexin and calreticulin. Biochemistry 37(10): 3480-90. 9521669

Wanamaker, C. P. and Green, W. N. (2005). N-linked glycosylation is required for nicotinic receptor assembly but not for subunit associations with calnexin. J. Biol. Chem. 280(40): 33800-10. 16091366

Wang, J. and White, A. L. (2000). Role of calnexin, calreticulin, and endoplasmic reticulum mannosidase I in apolipoprotein(a) intracellular targeting. Biochemistry 39: 8993-9000. 10913312

Ware, F. E., et al. (1995). The molecular chaperone calnexin binds Glc1Man9GlcNAc2 oligosaccharide as an initial step in recognizing unfolded glycoproteins. J. Biol. Chem. 270: 4697-4704. 7876241

Wilson, C. M., Farmery, M. R. and Bulleid, N. J. (2000). Pivotal role of calnexin and mannose trimming in regulating the endoplasmic reticulum-associated degradation of major histocompatibility complex class I heavy chain. J. Biol. Chem. 275: 21224-21432. 10801790


Biological Overview

date revised: 20 May 2006

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