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Query: UNIPROT:P06889 (Mol)
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Several biochemical properties of a variant surface glycoprotein (VSG) from the parasite Trypanosoma (Duttonella) vivax have been determined. ILDat 2.1 VSG is approximately 40 kDa in size making this the smallest trypanosome VSG described to date. The glycolipid anchor of ILDat 2.1 VSG is resistant to treatment with T. brucei-derived phospholipase C and data based on lectin affinity chromatography, incorporation of radiolabelled sugar and treatment with endoglycosidase H suggest that the T. vivax VSG bears little carbohydrate. cDNA to ILDat 2.1 VSG mRNA has been cloned and the encoded protein sequence includes the N-terminal amino acid peptide sequence derived from native VSG. The molecular weight of the VSG predicted from the translated cDNA sequence is similar to that of the native molecule and in support of the biochemical data it is devoid of sites for N-linked glycosylation. Examination of the deduced ILDat 2.1 VSG protein sequence reveals that it is most similar to T. congolense VSGs in the distribution of Cys residues and like the former it does not contain any of the defined VSG C-terminal domain types. However, unlike T. congolense VSGs it does not readily fit into the currently described VSG N-terminal domain types. Our studies suggest that ILDat 2.1 VSG is distinct from any of the previously characterized VSGs.
Mol Biochem Parasitol 1996 Nov 12
PMID:Characterization of a small variable surface glycoprotein from Trypanosoma vivax. 894 46

The structures of N-linked oligosaccharides from various Leishmania life-cycle stages and species have been investigated in order to elucidate differences which may be correlated with virulence or tissue tropisms. The structure of gp63 glycans from L major log- and stationary-phase promastigotes were elucidated and compared with the total membrane associated oligosaccharides from five Leishmania spp. L. major gp63 glycans from promastigotes in either log or stationary phases of their growth cycle were shown to have two neutral oligosaccharides having Bio-Gel P4 hydrodynamic volumes of 10.5 and 9.6 glucose units (GU). Sequential exoglycosidase digestion, fragmentation by acetolysis and methylation analysis of hydrazine released glycans, revealed the structure of G9.6 to be a biantennary oligomannose type, having the composition Man6GlcNAc2. These data were confirmed by structural analysis of gp63 oligosaccharides released by digestion with endo-beta-N-acetylglucosaminidase H (Endo-H) and N-glycanase F. The larger glycan was found to be terminally glucosylated, having the composition GlcMan6GlcNAc2. These oligosaccharides were found to occupy only two of the three predicted N-linked glycosylation sites in the L. major gp63 molecule, at positions 300 and 407. On comparison with glycans from other Leishmania spp. and strains, these two oligosaccharides were consistently found to be the predominant promastigote structures. Following transformation to the amastigote stage, alterations in N-linked oligosaccharides appeared to be less consistent between species. L. m. mexicana amastigotes were found to display the same G10.5 and G9.6 glycans found on promastigotes while L. donovani LV9 amastigotes were found to be devoid of N-linked glycans.
Mol Biochem Parasitol 1997 Jan
PMID:A unique, terminally glucosylated oligosaccharide is a common feature on Leishmania cell surfaces. 904 19

Function and biochemical properties of the V2 vasopressin receptor (V2R) mutant R337ter, identified in patients suffering from X-linked recessive nephrogenic diabetes insipidus, were investigated by expression in COS.M6 or HEK293 cells. Binding assays and measurements of adenylyl cyclase activity failed to detect function for the truncated receptor, although metabolic labeling demonstrated normal levels of protein synthesis. ELISA assays performed on cells expressing the receptors tagged at the amino terminus with the HA epitope failed to detect V2R R337ter on the plasma membrane. Treatment with endoglycosidase H revealed that the receptor was present only as a precursor form because the mature R337ter V2R, resistant to endoglycosidase H treatment, was not detected. The precursor of V2R-R337ter had a longer half-life than that of the wild type V2R, suggesting that arrested maturation may slow the degradation of the precursor. Unrelated experiments had demonstrated that V2R-G345ter, containing eight additional amino acids, was expressed on the plasma membrane and functioned normally. Receptor truncations longer than 337ter revealed that four of the eight amino acids identified initially provided the minimum length required for the protein to acquire cell surface expression. This was shown by the production of mature receptor (V2R-341ter) detectable in SDS-PAGE, which mediated arginine vasopressin stimulation of adenylyl cyclase activity and bound ligand. In addition, the identity of amino acid 340 was found to play a role in this phenomenon. In conclusion, these data demonstrate that the V2R R337ter is nonfunctional because it does not reach the plasma membrane and that the minimal protein length required for translocation of the V2R to the cell surface is sufficient to confer function to the receptor protein. They also suggest the existence of a protein quality control in the endoplasmic reticulum independent of glycosylation.
Mol Endocrinol 1997 Jun
PMID:An X-linked NDI mutation reveals a requirement for cell surface V2R expression. 917 Dec 34

Calsequestrin (CSQ), the major low-affinity Ca(2+)-binding glycoprotein of striated muscle fibers, is concentrated to yield aggregates that occupy the lumen of the terminal cisternae of the sarcoplasmic reticulum (SR). When infected or transfected into L6 myoblast, the protein is also concentrated, however, in dense vacuoles apparently separate from the endoplasmic reticulum (ER). CSQ-rich cells appear otherwise normal; in particular, neither other proteins involved in Ca2+ homeostasis nor ER chaperones are increased. The CSQ dense vacuoles are shown herein to be specialized ER subdomains as demonstrated by 1) the endoglycosidase H sensitivity of their CSQ and 2) two markers, calreticulin and calnexin (but not others, protein disulfide isomerase and BiP), intermixed with the vacuole content. Their formation is shown to start with the aggregation of CSQ at discrete sites of the ER lumen. When cells were transfected with both CSQ and calreticulin, only the first gave rise to vacuoles; the second remained diffusely distributed within the ER lumen. The possibility that CSQ aggregation is an artifact of overexpression appears unlikely because 1) within dense vacuoles CSQ molecules are not disulfide cross-linked, 2) their turnover is relatively slow (t = 12 h), and 3) segregated CSQ is bound to large amounts of Ca2+. Transfection of a tagged CSQ into cells already overexpressing the protein revealed the continuous import of the newly synthesized protein into preassembled vacuoles. The tendency to aggregation appears, therefore, as a property contributing to the segregation of CSQ within the ER lumen and to its accumulation within specialized subdomains. The study of L6 cells expressing CSQ-rich vacuoles might thus ultimately help to unravel mechanisms by which the complexity of the sarcoplasmic reticulum is established in muscle fibers.
Mol Biol Cell 1997 Sep
PMID:Overexpression of calsequestrin in L6 myoblasts: formation of endoplasmic reticulum subdomains and their evolution into discrete vacuoles where aggregates of the protein are specifically accumulated. 930 74

The biogenesis of peptide hormone secretory granules involves a series of sorting, modification, and trafficking steps that initiate in the trans-Golgi and trans-Golgi network (TGN). To investigate their temporal order and interrelationships, we have developed a pulse-chase protocol that follows the synthesis and packaging of a sulfated hormone, pro-opiomelanocortin (POMC). In AtT-20 cells, sulfate is incorporated into POMC predominantly on N-linked endoglycosidase H-resistant oligosaccharides. Subcellular fractionation and pharmacological studies confirm that this sulfation occurs at the trans-Golgi/TGN. Subsequent to sulfation, POMC undergoes a number of molecular events before final storage in dense-core granules. The first step involves the transfer of POMC from the sulfation compartment to a processing compartment (immature secretory granules, ISGs): Inhibiting export of pulse-labeled POMC by brefeldin A (BFA) or a 20 degrees C block prevents its proteolytic conversion to mature adrenocorticotropic hormone. Proteolytic cleavage products were found in vesicular fractions corresponding to ISGs, suggesting that the processing machinery is not appreciably activated until POMC exits the sulfation compartment. A large portion of the labeled hormone is secreted from ISGs as incompletely processed intermediates. This unregulated secretory process occurs only during a limited time window: Granules that have matured for 2 to 3 h exhibit very little unregulated release, as evidenced by the efficient storage of the 15-kDa N-terminal fragment that is generated by a relatively late cleavage event within the maturing granule. The second step of granule biogenesis thus involves two maturation events: proteolytic activation of POMC in ISGs and a transition of the organelle from a state of high unregulated release to one that favors intracellular storage. By using BFA, we show that the two processes occurring in ISGs may be uncoupled: although the unregulated secretion from ISGs is impaired by BFA, proteolytic processing of POMC within this organelle proceeds unaffected. The finding that BFA impairs constitutive secretion from both the TGN and ISGs also suggests that these secretory processes may be related in mechanism. Finally, our data indicate that the unusually high levels of unregulated secretion often associated with endocrine tumors may result, at least in part, from inefficient storage of secretory products at the level of ISGs.
Mol Biol Cell 1997 Nov
PMID:Distinct molecular events during secretory granule biogenesis revealed by sensitivities to brefeldin A. 936 61

The juvenile hormone analog (JHA) methoprene was used to induce the synthesis of the yolk protein precursor vitellogenin (Vg) in adult females and males of the cockroach Leucophaea maderae. The female- and male-produced vitellogenin (VgF and VgM, respectively) contained polypeptides of 112, 95, 92, and 54 kDa. Also present in the secreted vitellogenins was a soffmall quantity of a short-lived transitional 155 kDa Vg polypeptide, and a variable amount of an 85 dDa species. Quantitatively, the VgF and VgM were significantly different in the Vg112 and Vg95 units (VgF > VgM), and in the Vg85 polypeptide (VgF < VgM). In the present study, the biosynthesis of Vg precursors in the fat bodies of females and males was examined using a short radiopulse with 35S-methionine/cysteine and 32P-orthophosphate. The glycosylation of the Vg precursors was examined by digestion with endoglycosidase H and by the inhibition of N-linked glycosylation with tunicamycin. The data showed that in both females and males, the synthesis of the vitellogenin precursor occurred in a stepwise fashion: (1) the co-translational glycosylation of Vg203; (2) the post-translational phosphorylation of Vg203 to form Vg220; (3) the proteolytic processing of Vg220 to form the constituent Vg polypeptides. The 203 and 220 kDa Vg precursors of females and males appeared to be similarly glycosylated and phosphorylated. The additional processing of Vg112 to Vg85 was more pronounced in the fat bodies of males than in females, and appears to account for the quantitative difference in the distribution of these polypeptides in VgF and VgM. Finally, the major oligosaccharides of VgF and VgM appear to be those of N-linked mannose residues. The treatment of females and males with tunicamycin indicated that the co-translational glycosylation of Vg precursors was required for the phosphorylation of the Vg precursor, as well as the secretion of Vg from the fat body.
Insect Biochem Mol Biol 1997 Nov
PMID:The biosynthesis and processing of vitellogenin in the fat bodies of females and males of the cockroach Leucophaea maderae. 953 60

To study the role of carbohydrate in lysosomal protein transport, we engineered two novel glycosylation signals (Asn-X-Ser/Thr) into the cDNA of human procathepsin L, a lysosomal acid protease. We constructed six mutant cDNAs encoding glycosylation signals at mutant sites Asn-138, Asn-175, or both sites together, in the presence or absence of the wild-type Asn-204 site. We stably transfected wild-type and mutant cDNAs into NIH3T3 mouse fibroblasts and then used species-specific antibodies to determine the glycosylation status, phosphorylation, localization, and transport kinetics of recombinant human procathepsin L containing one, two, or three glycosylation sites. Both novel glycosylation sites were capable of being glycosylated, although Asn-175 was utilized only 30-50% of the time. Like the wild-type glycosylation at Asn-204, carbohydrates at Asn-138 and Asn-175 were completely sensitive to endoglycosidase H, and they were phosphorylated. Mutant proteins containing two carbohydrates were capable of being delivered to lysosomes, but there was not a consistent relationship between the efficiency of lysosomal delivery and carbohydrate content of the protein. Pulse-chase labeling revealed a unique biosynthetic pattern for proteins carrying the Asn-175 glycosylation sequence. Whereas wild-type procathepsin L and mutants bearing carbohydrate at Asn-138 appeared in lysosomes by about 60 min, proteins with carbohydrate at Asn-175 were processed to a lysosome-like polypeptide within 15 min. Temperature shift, brefeldin A, and NH4Cl experiments suggested that the rapid processing did not occur in the endoplasmic reticulum and that Asn-175 mutants could interact with the mannose 6-phosphate receptor. Taken together, our results are consistent with the interpretation that Asn-175 carbohydrate confers rapid transport to lysosomes. We may have identified a recognition domain in procathepsin L that is important for its interactions with the cellular transport machinery.
Mol Biol Cell 1998 May
PMID:Effect of carbohydrate position on lysosomal transport of procathepsin L. 957 Dec 45

The tailpiece of secretory Ig-mu-chains (mu(s)tp) is highly conserved throughout evolution: in particular, a carboxy-terminal cysteine residue (Cys575) and a glycan linked to Asn563 are found in all species sequenced so far. Here we show that the mu(s)tp oligosaccharide moieties are important for the binding of J-chains and for the process of IgM polymerization. In the absence of the mu(s)tp glycans, pentamers cannot be assembled and polymers containing six or more subunits are secreted. Despite their increased valency, these molecules have a lower association rate with antigen than wild-type polymers. Unexpectedly, the C-terminal oligosaccharides also affect kinetic parameters on unpolymerized subunits. Thus, monomers lacking the C-terminal sugars because of either site-directed mutagenesis or selective enzymatic deglycosylation with endoglycosidase H, have a lower k(on) for the antigen. Taken together, our results indicate that the C-terminal mu-chain glycans can shape the structure of mu(s2)L2 subunits and their further assembly into polymers.
Mol Immunol 1998 Sep
PMID:Biogenesis and function of IgM: the role of the conserved mu-chain tailpiece glycans. 983 52

Trypanosoma cruzi is a protozoan parasite that belongs to an early branch in evolution. Although it lacks several features of the pathway of protein N-glycosylation and oligosaccharide processing present in the endoplasmic reticulum of higher eukaryotes, it displays UDP-Glc:glycoprotein glucosyltransferase and glucosidase II activities. It is herewith reported that this protozoan also expresses a calreticulin-like molecule, the third component of the quality control of glycoprotein folding. No calnexin-encoding gene was detected. Recombinant T. cruzi calreticulin specifically recognized free monoglucosylated high-mannose-type oligosaccharides. Addition of anti-calreticulin serum to extracts obtained from cells pulse-chased with [35S]Met plus [35S]Cys immunoprecipitated two proteins that were identified as calreticulin and the lysosomal proteinase cruzipain (a major soluble glycoprotein). The latter but not the former protein disappeared from immunoprecipitates upon chasing cells. Contrary to what happens in mammalian cells, addition of the glucosidase II inhibitor 1-deoxynojirimycin promoted calreticulin-cruzipain interaction. This result is consistent with the known pathway of protein N-glycosylation and oligosaccharide processing occurring in T. cruzi. A treatment of the calreticulin-cruzipain complexes with endo-beta-N-acetylglucosaminidase H either before or after addition of anti-calreticulin serum completely disrupted calreticulin-cruzipain interaction. In addition, mature monoglucosylated but not unglucosylated cruzipain isolated from lysosomes was found to interact with recombinant calreticulin. It was concluded that the quality control of glycoprotein folding appeared early in evolution, and that T. cruzi calreticulin binds monoglucosylated oligosaccharides but not the protein moiety of cruzipain. Furthermore, evidence is presented indicating that glucosyltransferase glucosylated cruzipain at its last folding stages.
Mol Biol Cell 1999 May
PMID:Trypanosoma cruzi calreticulin is a lectin that binds monoglucosylated oligosaccharides but not protein moieties of glycoproteins. 1023 51

Variant splicing of gonadotropin receptor mRNA commonly occurs, however expression of receptor protein variants and their trafficking has yet to be studied in detail. To determine receptor variant trafficking and intracellular processing in mammalian cells, the intracellular fate of intentionally truncated variants of human follicle stimulating hormone receptor (hFSH-R) expressed in CHO cells was examined. Monoclonal antibodies (mAbs) were made against the hFSH-R's extracellular domain (ECD) expressed in insect cells. Four mAbs 106.156, 106.290, 106.318, and 106.263 were chosen as probes. Epitope mapping using synthetic peptides, and truncated hFSH-R variants revealed that mAb 106.156 bound to ECD residues 183-220, while mAbs 106.318, 106.290, 106.263 bound ECD residues 300-331. Immunofluorescence microscopy showed that mAbs 106.318 and 106.156 stained the surface of fixed, intact CHO cells expressing wild type hFSH-R. However, following cell permeabilization all four antibodies stained hFSH-R in Golgi and endoplasmic reticulum. Permeabilized cells expressing truncated variants ECD213 and ECD254 showed staining accumulated in the endoplasmic reticulum/nuclear envelope continuum. ECD335/His was found to accumulate in extended endoplasmic reticulum (ER). The ER location of ECD335/His was confirmed by double labeling experiments with concanavalin A and ECD mAb. Glycosidase digestion followed by Western blot analysis show ECD213 and ECD335/His to be glycosylated, but not ECD254. Both glycosylated truncated hFSH-R variants were sensitive to peptide-N-glycanase F and endoglycosidase H but insensitive to neuraminidase indicating that these variants possess high mannose type oligosaccharides. Thus truncated hFSH-R variants do not reach the medial or trans Golgi where high mannose oligosaccharides are trimmed and sialic acid is added. These data suggest that the conformation the ECD of the wild type receptor is different from the ECD alone expressed in the endoplasmic reticulum. This information suggests that the ECD serves two distinct roles; the first is to bind FSH and the other is likely to contact the endodomain of the receptor, which presumably leads to activation of the endodomain for signal transduction.
Mol Cell Endocrinol 2000 Feb 25
PMID:Human follicle stimulating hormone receptor variants lacking transmembrane domains display altered post-translational conformations. 1071 54


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