Distinct roles of endoplasmic reticulum cytochrome b5 and fused cytochrome b5-like domain for rat Delta6-desaturase activity

The Delta6-desaturase catalyzes key steps in long-chain polyunsaturated fatty acid biosynthesis. Although the gene coding for this enzyme has been isolated in diverse animal species, the protein structure remains poorly characterized. In this work, rat Delta6-desaturase expressed in COS-7 cells was shown to localize in the endoplasmic reticulum. As the enzyme contains an N-terminal cytochrome b5-like domain, we investigated by site-directed mutagenesis the role of this domain in the enzyme activity. The typical HPGG motif of the cytochrome b5-like domain, and particularly histidine in this motif, is required for the activity of the enzyme, whatever the substrate. Neither endogenous COS-7 cytochrome b5 nor coexpressed rat endoplasmic reticulum cytochrome b5 could rescue the activity of mutated forms of Delta6-desaturase. Moreover, when rat endoplasmic reticulum cytochrome b5 was coexpressed with wild-type desaturase, both proteins interacted and Delta6-desaturase activity was significantly increased. The identified interaction between these proteins is not dependent on the desaturase HPGG motif. These data suggest distinct and essential roles for both the desaturase cytochrome b5-like domain and free endoplasmic reticulum cytochrome b5 for Delta6-desaturase activity.     

Glycosylation of the hepatitis C virus envelope protein E1 occurs posttranslationally in a mannosylphosphoryldolichol-deficient CHO mutant cell line

The addition of N-linked glycans to a protein is catalyzed by oligosaccharyltransferase, an enzyme closely associated with the translocon. N-glycans are believed to be transferred as the protein is being synthesized and cotranslationally translocated in the lumen of the endoplasmic reticulum. We used a mannosylphosphoryldolichol-deficient Chinese hamster ovary mutant cell line (B3F7 cells) to study the temporal regulation of N-linked core glycosylation of hepatitis C virus envelope protein E1. In this cell line, truncated Glc(3)Man(5)GlcNAc(2) oligosaccharides are transferred onto nascent proteins. Pulse-chase analyses of E1 expressed in B3F7 cells show that the N-glycosylation sites of E1 are slowly occupied until up to 1 h after protein translation is completed. This posttranslational glycosylation of E1 indicates that the oligosaccharyltransferase has access to this protein in the lumen of the endoplasmic reticulum for at least 1 h after translation is completed. Comparisons with the N-glycosylation of other proteins expressed in B3F7 cells indicate that the posttranslational glycosylation of E1 is likely due to specific folding features of this acceptor protein.     

Mammalian plasma membrane ecto-nucleoside triphosphate diphosphohydrolase 1, CD39, is not active intracellularly. The N-glycosylation state of CD39 correlates with surface activity and localization

CD39 is a member of the membrane-bound ecto-nucleoside triphosphate diphosphohydrolase family. The active site for native CD39 is located on the outer surface of the cellular plasma membrane; however, it is not yet known at what stage this enzyme becomes active along the secretory pathway to the plasma membrane. In this study, sucrose density fractionations performed on CD39-transfected COS-7 cell membranes suggest that CD39 activity resides primarily in the plasma membrane. Furthermore, we have created recombinant, soluble versions of CD39, one that is secreted and others that are retained in the endoplasmic reticulum, to demonstrate that CD39 is not active until it reaches the plasma membrane both in yeast and COS-7 cells. Moreover, the secreted active soluble CD39 in COS-7 cells is found to receive a higher degree of N-glycan addition than the inactive form retained intracellularly. When COS-7 cells were treated with tunicamycin to prevent N-glycosylation, soluble CD39 was not detected in the extracellular medium and remained inactive intracellularly. Surface biotinylation analysis also revealed that surface-expressed wild type CD39 receives a higher degree of N-glycosylation than intracellular forms and that inhibition of N-glycosylation prevents its plasma membrane localization. In addition, both intact and digitonin-permeablized COS-7 cells transfected with CD39 possess similar ecto-ATPase activities, further supporting the conclusion that only surface-expressed CD39 is enzymatically active. All of these data suggest that intracellular CD39 is inactive and that only a fully glycosylated CD39 has apyrase activity and is localized at the cell surface.     

Cleavage of calnexin caused by apoptotic stimuli: implication for the regulation of apoptosis

Calnexin is an endoplasmic reticulum (ER)-resident molecular chaperone that plays an essential role in the correct folding of membrane proteins. We found that calnexin is subjected to partial cleavage in apoptotic mouse cells. Both ER stress-inducing and ER stress-non-inducing apoptotic stimuli caused the cleavage of calnexin, indicating that this event does not always occur downstream of ER stress. The inhibition of caspases that target the amino acid sequence DXXD abrogated calnexin cleavage in apoptotic stimulus-treated cells. In addition, disruption of one of two DXXD sequences located in the cytoplasmic domain caused calnexin to escape cleavage during apoptosis. Furthermore, calnexin was cleaved in vitro by recombinant caspase-3 or caspase-7. Finally, the overexpression of a presumed cleavage product of calnexin partly inhibited apoptosis. These results collectively suggest that caspase-3 or caspase-7 cleaves calnexin, whose cleaved product leads to the attenuation of apoptosis.     

Cell surface expression of calnexin, a molecular chaperone in the endoplasmic reticulum

The folding and assembly of nascent proteins in the endoplasmic reticulum are assisted by the molecular chaperone calnexin, which is itself retained within the endoplasmic reticulum. It was up to now assumed that calnexin was selectively expressed on the surface of immature thymocytes because of a particular characteristic of the protein sorting machinery in these cells. We now report that a small fraction of calnexin is normally expressed on the surface of various cells such as mastocytoma cells, murine splenocytes, fibroblast cells, and human HeLa cells. Surface biotinylation followed by chase culture of living cells revealed that calnexin is continuously delivered to the cell surface and then internalized for lysosomal degradation. These results suggest that there is continuous exocytosis and endocytosis of calnexin, and the amount of calnexin on the plasma membrane results from the balance of the rates of these two events. To study the structural requirement of calnexin for surface expression, we created deletion mutants of calnexin and found that the luminal domain, particularly the glycoprotein binding domain, is necessary. These findings suggest that the surface expression of calnexin depends on the association with glycoproteins and that calnexin may play a certain role as a chaperone on the plasma membrane as well.     

ER Reorganization is Remarkably Induced in COS-7 Cells Accumulating Transmembrane Protein Receptors Not Competent for Export from the Endoplasmic Reticulum

The newly synthesized mutant L501fsX533 Frizzled-4 form and the alpha3beta4 nicotinic acetylcholine receptor expressed in the absence of nicotine accumulate in the endoplasmic reticulum of COS-7 cells and induce the formation of large areas of smooth and highly convoluted cisternae. This results in a generalized block of the transport to the Golgi complex of newly synthesized proteins. Intriguingly, both effects happen peculiarly in COS-7 cells; HeLa, Huh-7, and HEK293 cells expressing the two receptors at similar level than COS-7 cells show normal ER and normal transport toward the plasma membrane. These results question the conclusion that a dominant-negative mechanism would explain the dominance of the mutant L501fsX533 Fz4 allele in the transmission of a form of Familial exudative vitreoretinopathy. Moreover, they indicate that the coordination of endoplasmic reticulum homeostasis in COS-7 cells is particularly error prone. This finding suggests that COS-7 cells may be extremely useful to study the molecular mechanisms regulating endoplasmic reticulum size and architecture.     

Cloning, expression, and characterization of an alternatively spliced variant of human heparanase

Heparanase is an endoglycosidase that cleaves heparan sulfate in the extracellular matrix (ECM) and hence participates in ECM degradation and remodeling. Heparanase is involved in fundamental biological processes such as cancer metastasis, angiogenesis, and inflammation. Alternative splicing in the coding region of human heparanase was not reported. Here, we report the cloning of a splice variant of human heparanase that lacks exon 5 and is missing 174 bp compared to the wild-type cDNA. Splice 5 is expressed as a 55 kDa protein compared to the 65 and 50 kDa latent and active wild-type enzyme. Splice 5 was not detected in the incubation medium of tumor cells as opposed to the wild-type latent heparanase. Splice 5 escaped proteolytic cleavage, was devoid of HS degradation activity and exhibited diffused rather than granular cellular localization.     

Calreticulin and calnexin in the endoplasmic reticulum are important for phagocytosis.

Calreticulin and calnexin are Ca2+-binding proteins with chaperone activity in the endoplasmic reticulum. These proteins have been eliminated by gene replacement in Dictyostelium, the only microorganism known to harbor both proteins; family members in Dictyostelium are located at the base of phylogenetic trees. A dramatic decline in the rate of phagocytosis was observed in double mutants lacking calreticulin and calnexin, whereas only mild changes occurred in single mutants. Dictyostelium cells are professional phagocytes, capable of internalizing particles by a sequence of activities: adhesion of the particle to the cell surface, actin-dependent outgrowth of a phagocytic cup, and separation of the phagosome from the plasma membrane. In the double-null mutants, particles still adhered to the cell surface, but the outgrowth of phagocytic cups was compromised. Green fluorescent protein-tagged calreticulin and calnexin, expressed in wild-type cells, revealed a direct link of the endoplasmic reticulum to the phagocytic cup enclosing a particle, such that the Ca2+ storage capacity of calreticulin and calnexin might directly modulate activities of the actin system during particle uptake.     

Cell-surface expression of a new splice variant of the mouse signal peptide peptidase

The signal peptide peptidase (SPP) is an intramembrane-cleaving aspartyl protease that acts on type II transmembrane proteins. SPP substrates include signal peptides after they have been cleaved from a preprotein, hence the name. The known SPP isoform, which we renamed SPPalpha, contains an endoplasmic reticulum retention signal at the carboxy terminus. We found a new splice variant, SPPbeta, with an additional in-frame exon inserted between exons 11 and 12 of SPPalpha. Insertion of the new exon led to a complete change in the amino-acid sequence of the carboxy tail. A stop codon within this new exon resulted in silencing of exon 12 and eliminated the endoplasmic reticulum retention signal. The new SPP isoform predominantly localised to the cell surface in contrast to the more restricted localisation of SPPalpha in the endoplasmic reticulum. Differential expression in mouse tissues and in subcellular compartments suggests new functions for SPP in addition to cleaving signal peptides.     

T cell receptor assembly and expression in the absence of calnexin

Most subunits of the alphabeta deltaepsilon gammaepsilon zetazeta T cell antigen receptor (TCR) complex associate with the molecular chaperone calnexin shortly after their synthesis in the endoplasmic reticulum, including clonotypic TCRalpha,beta molecules and invariant CD3gamma,delta,epsilon chains. While calnexin interaction is suggested to be important for the stability of newly synthesized TCRalpha subunits, the role of calnexin in the survival and assembly of remaining TCR components is unknown. Here we evaluated the expression of TCR proteins in CEM T cells and the calnexin-deficient CEM variant CEM.NK(R). We found that CEM and CEM.NK(R) cells constitutively synthesized all TCR subunits except for TCRalpha and that CD3gamma,delta,epsilon components and CD3-beta complexes were effectively assembled together in both cell types. The stability and folding of core CD3epsilon chains were similar in CEM and CEM.NK(R) cells. Interestingly, TCRalpha synthesis was differentially induced by phorbol myristate acetate treatment in CEM and CEM.NK(R) cells and TCRalpha proteins synthesized in CEM.NK(R) cells showed reduced survival compared to those made in CEM cells. Importantly, these data show that TCR complexes were inducibly expressed on CEM.NK(R) cells in the absence of calnexin synthesis. These results demonstrate that TCR complexes can be expressed in the absence of calnexin and suggest that the role of calnexin in the quality control of TCR assembly is primarily restricted to the stabilization of newly synthesized TCRalpha proteins.     

Chondroitin Sulfate Synthase-2/Chondroitin Polymerizing Factor Has Two Variants with Distinct Function

Chondroitin sulfate (CS) is a polysaccharide consisting of repeating disaccharide units of N-acetyl-d-galactosamine and d-glucuronic acid residues, modified with sulfated residues at various positions. To date six glycosyltransferases for chondroitin synthesis have been identified, and the complex of chondroitin sulfate synthase-1 (CSS1)/chondroitin synthase-1 (ChSy-1) and chondroitin sulfate synthase-2 (CSS2)/chondroitin polymerizing factor is assumed to play a major role in CS biosynthesis. We found an alternative splice variant of mouse CSS2 in a data base that lacks the N-terminal transmembrane domain, contrasting to the original CSS2. Here, we investigated the roles of CSS2 variants. Both the original enzyme and the splice variant, designated CSS2A and CSS2B, respectively, were expressed at different levels and ratios in tissues. Western blot analysis of cultured mouse embryonic fibroblasts confirmed that both enzymes were actually synthesized as proteins and were localized in both the endoplasmic reticulum and the Golgi apparatus. Pulldown assays revealed that either of CSS2A, CSS2B, and CSS1/ChSy-1 heterogeneously and homogeneously interacts with each other, suggesting that they form a complex of multimers. In vitro glycosyltransferase assays demonstrated a reduced glucuronyltransferase activity in CSS2B and no polymerizing activity in CSS2B co-expressed with CSS1, in contrast to CSS2A co-expressed with CSS1. Radiolabeling analysis of cultured COS-7 cells overexpressing each variant revealed that, whereas CSS2A facilitated CS biosynthesis, CSS2B inhibited it. Molecular modeling of CSS2A and CSS2B provided support for their properties. These findings, implicating regulation of CS chain polymerization by CSS2 variants, provide insight in elucidating the mechanisms of CS biosynthesis.     

Osteopontin-deficient bone cells are defective in their ability to produce NO in response to pulsatile fluid flow

We have previously identified a mutation (R273W) in the von Willebrand factor (VWF) propeptide that results in quantitative deficiency of plasma VWF and a loss of high molecular weight VWF multimers. Recombinant VWF having the R273W mutation (rVWFR273W) expressed in COS-7 cells demonstrated severely impaired secretion and degradation in an intracellular location [Allen, S., et al. (2000) Blood 96, 560-568]. In this report we used pulse-chase analysis and endoglycosidase H digestion of wild-type rVWF and rVWFR273W immunoprecipitated from COS-7 cells to show that rVWFR273W was retained in the endoplasmic reticulum (ER). We demonstrate for the first time that wild-type rVWF and rVWFR273W interacted with the thiol-dependent oxidoreductase ERp57 during biosynthesis in the ER. Pulse chase analysis demonstrated that the interactions of rVWFR273W with ERp57 and calnexin were prolonged compared to wild-type rVWF. In contrast there was no apparent difference between rVWFR273W and wild-type rVWF in their time-courses of interaction with calreticulin.