Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P06889 (Mol)
 630,302 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

AtT-20 cells contain two molecular weight forms (87 and 66 kDa) of the prohormone convertase PC1 (also known as PC3), thought to be involved in prohormone maturation. In this study we found that PC1 is first synthesized as a 94-kDa protein, which is then rapidly converted to a 84-kDa form. Two lines of evidence suggest that the generation of the 84-kDa protein from its 94-kDa precursor occurs in the endoplasmic reticulum (ER). The processing of the 94-kDa protein to the lower molecular weight form was extremely rapid, occurring with a half-life less than 2 min. The 84-kDa form was initially endoglycosidase H-sensitive, indicating lack of acquisition of sugars transferred in the medial Golgi. Within 40 min after the labeling period, the 84-kDa protein was converted to an endoglycosidase H-resistant form of 87 kDa, which was then processed to an endoglycosidase H-resistant 66-kDa protein. Radiosequencing of the 87- and 66-kDa proteins indicated that the biosynthesis of the 87-kDa protein involves the removal of the 83 amino acid Pro segment and that the processing of the 87-kDa to the 66-kDa form occurred by cleavage of a carboxy-terminal segment. Brefeldin A did not interrupt the cleavage of the 94-kDa to the 87-kDa protein, but completely blocked the processing of the 84- to 87-kDa proteins to the 66-kDa species. The 84-kDa protein produced in brefeldin-treated cells remained sensitive to endoglycosidase H, indicating a lack of exposure to Golgi sugar transferases.(ABSTRACT TRUNCATED AT 250 WORDS)
Mol Cell Neurosci 1994 Jun
PMID:Evidence for cleavage of the PC1/PC3 pro-segment in the endoplasmic reticulum. 808 24

A recombinant lambda phage was identified in a Clostridium perfringens genomic library by means of its ability to hydrolyse the fluorescent substrate 4-methyl-umbelliferyl-beta-D-glucosaminide, isolated and shown to encode an endo-beta-N-acetylglucosaminidase. This enzyme, NagH, is also known as hyaluronidase, or Mu toxin, a putative virulence factor which is likely to act on connective tissue during gas gangrene. Nucleotide sequence analysis allowed the primary structure to be deduced and showed hyaluronidase to be a large exported protein of 114,392 Daltons and an enzyme of this size, endowed with the corresponding activities, was partially purified from C. perfringens. Hyaluronidase seems to be organised into two domains, an N-terminal region comprising 700 amino acids bearing the active site and a 300-residue C-terminal segment, containing three copies of an extended motif. Two other reading frames, linked to nagH, also appear to encode proteins with sugar-binding motifs.
Mol Gen Genet 1994 Apr
PMID:Molecular genetic analysis of the nagH gene encoding a hyaluronidase of Clostridium perfringens. 817 18

The aspartic proteinase (MPP) gene from the zygomycete fungus Mucor pusillus was introduced into an ascomycete fungus, Aspergillus oryzae, by protoplast transformation using the nitrate reductase (niaD) gene as the selective marker. Southern blot analysis indicated that the MPP gene was integrated into the resident niaD locus at a copy number of 1-2. MPP secreted by the recombinant A. oryzae was correctly processed but was more highly glycosylated than that produced in the original M. pusillus strain. Treatment with endo-beta-N-acetylglucosaminidase H and analysis of the carbohydrate composition of the secreted MPP revealed that the extra glycosylation of the MPP secreted by the recombinant A. oryzae was due to altered processing of mannose residues. The extra glycosylation of MPP affected its enzyme properties including its milk-clotting and proteolytic activities.
Mol Gen Genet 1993 Nov
PMID:Characterization of an aspartic proteinase of Mucor pusillus expressed in Aspergillus oryzae. 824 85

To investigate how class II major histocompatibility complex (MHC) molecules are released from complexes with invariant chain (Ii), we studied a 25 kDa Ii fragment (p25) detected by Western blotting in affinity chromatographed DR preparations. The p25 species corresponds to the non-transmembrane, C-terminal Ii fragment 107-232. It was determined by gel filtration chromatography that the p25 fragment has a relative molecular mass (M(r)) of 46 kDa, indicating that this Ii fragment is present as dimers in B cell lysate. Two independent approaches were followed to demonstrate that generation of the p25 fragment takes place shortly before, or concomitantly to, loading of class II MHC molecules with antigen fragments. First, it was shown that a fraction of the p25 molecules is resistant to endoglycosidase H digestion, indicating that the p25 polypeptide can exit the endoplasmic reticulum (ER) and is transported at least to the cis-Golgi compartment. Second, treatment of class II MHC-positive B cells with leupeptin blocks the formation of p25, further indicating that this Ii fragment is generated in the endosomal compartment. The role of the p25 Ii species in the assembly of complexes between peptides and DR molecules was then investigated. While the p25 fragment was totally unable to prevent binding of a synthetic tetanus toxin peptide to DR molecules, the full-length Ii species (p33/35) effectively inhibited peptide binding, indicating that, by contrast with the p33/35 species, the p25 fragment does not occlude the peptide binding site of DR molecules. We concluded that the p25 fragment, which is produced by proteolytic cleavage at the N-terminal side of Methionine 107, has a decreased affinity for DR molecules as compared with the p33/35 species. Dissociation of the p25 fragment from DR molecules exposes the peptide binding site, which is thus made accessible for antigen fragments. This model of the complexes between DR and antigen fragments proposes that a stretch of Ii prevents peptide binding by occluding the peptide binding site without directly occupying it.
Mol Immunol 1993 Dec
PMID:Release of DR molecules from complexes with invariant chain through the formation of a C-terminal 25 kDa invariant chain fragment. 827 76

An endogenous Madin-Darby canine kidney (MDCK) lysosomal membrane glycoprotein that exhibits a basolateral targeting pathway to the lysosome is shown here to exhibit significant N-terminal amino acid sequence identity to lysosomal associated membrane proteins (LAMP-2) of other species. During establishment of the MDCK monolayer after only 1 d of culture, this canine LAMP-2 has a larger molecular size (110 kDa) than following formation of a confluent monolayer after 3 d of culture (100 kDa) due to the increased presence of N-linked polylactosamine oligosaccharide chains. Neither polylactosamine glycosylation of LAMP-2 in MDCK cells nor truncation of N-linked oligosaccharide chains of LAMP-2 in a ricin-resistant MDCK-RCAR cell line influenced the basolateral polarity of its targeting. However, the rate of basolateral delivery of LAMP-2 in MDCK cells plated for 3 d was significantly faster (t1/2 = 28 min) than in 1-d cells (t1/2 = 40 min); in MDCK-RCAR cells the rate of basolateral delivery at both 1 and 3 d of plating was similar (t1/2 = 40 min). The rate differential in MDCK cells occurred after arrival of LAMP-2 to the Golgi apparatus because the rate of acquisition of endoglycosidase H resistance was the same (t1/2 = 25 min) at both days of plating. The rate of transit of LAMP-2 through the Golgi apparatus to the basolateral domain was therefore far more rapid (approximately 4-fold) in 3 d compared with 1-d MDCK cultures. The increased polylactosamine glycosylation of MDCK LAMP-2 at early times of plating during the establishment of a confluent epithelial monolayer may thus be related to its longer residence time in the Golgi apparatus.
Mol Biol Cell 1993 Jun
PMID:Increased LAMP-2 polylactosamine glycosylation is associated with its slower Golgi transit during establishment of a polarized MDCK epithelial monolayer. 837 71

Intracellular cleavage of class II MHC-associated Ii to p21 and p10 and the appearance of Ii-freed alpha, beta dimers were concurrent events lasting from 1 to 6 hr after synthesis of alpha, beta, Ii trimers, possibly related to charging of foreign peptides to the class II MHC antigen-binding site. Sequential immunoprecipitations of pulse-chase radiolabeled cells were made four times with anti-Ii monoclonal antibody to remove Ii and alpha, beta, Ii trimers and then with anti-class II antibody to detect the time-dependent appearance of Ii-freed alpha, beta dimers. The cleavage of Ii to p21 and p10 was revealed in leupeptin-treated cells. Cell treatment with Brefeldin A (BFA) was associated with a decrease in Ii-freed alpha, beta dimers, with inhibition of leupeptin-revealed cleavage of Ii to p21 and p10, and with persistence of endoglycosidase H susceptibility of Ii and class II alpha, beta chains. We conclude that in untreated cells, cleavage and release of Ii from class II MHC alpha and beta chains occur after those complexes traverse a BFA-sensitive step in the Golgi apparatus.
Mol Immunol 1993 Feb
PMID:Effects of brefeldin A on cleavage of invariant chain to p21 and p10 and the appearance of Ii-freed class II MHC dimers. 842 32

This study was undertaken to investigate the nature and microheterogeneity of the carbohydrate moiety of the Fc epsilon receptors of RBL-CA10 and RBL-CA10.7 cells. Treatment using the glycosylation processing inhibitors, castanospermine (CN), 1-deoxymannojirimycin (DMJ), and swainsonine (SW) resulted in a decrease of the relative molecular mass (M(r)) of both the alpha-chain of the high affinity receptor for IgE, Fc epsilon RI(alpha), and the low affinity receptor for IgE, Fc epsilon RL. Exposure to DMJ had the greatest effect on the M(r), while CN seemed to lead to a decreased cell surface expression of Fc epsilon RI. Both receptors are largely resistant to endoglycosidase H as their M(r) decreased only by approximately 2 kDa. These results suggest that both receptors are composed primarily of complex oligosaccharides with a single high mannose, N-glycosylated site. Both Fc epsilon receptors become endoglycosidase H sensitive if first exposed to DMJ which indicates that the carbohydrate composition is indeed altered by this processing inhibitor presumably by blocking the formation of complex structures. When the Fc epsilon receptors were reduced and hydrolyzed by N-glycanase, the M(r) values for Fc epsilon RI(alpha) and Fc epsilon RL decreased to approximately 28 and 34-38 kDa respectively. In the case of Fc epsilon RI(alpha), this implies the presence of only a small amount of O-linked oligosaccharides.
Mol Immunol 1993 Feb
PMID:The N-linked oligosaccharides of the Fc epsilon receptors of rat basophilic leukemia cells. 843 10

It has recently been reported that Asp 397 of the rat lutropin/ choriogonadotropin receptor (rLHR) may be involved in transducing the signal from hormone binding to the stimulation of cAMP production. We examined the analogous region in the rat follitropin receptor (rFSHR) by substituting the Asp at position 404 (D404) of the rFSHR with either Glu (D404E), Ala (D404A), or Lys (D404K). Both in intact 293 cells and in detergent-solubilized extracts of 293 cells transiently transfected with the rFSHR constructs, only the wild type rFSHR exhibited detectable binding activity. Although the D404-substituted rFSHR mutants were visible on Western blots, in contrast to the wild type rFSHR which is present on Western blots as both mature and immature forms, only a single band comigrating with immature receptor was observed for the mutants. Furthermore, these mutants were sensitive to endoglycosidase H (Endo H), thus indicating that the mutant receptor proteins were retained intracellularly in the endoplasmic reticulum. To test whether the lack of binding of the D404-substituted rFSHR mutants was due to a perturbation of a binding site or to the intracellular retention of the mutants, a truncated rFSHR(t637) mutant, containing a cytoplasmic truncation that should not directly affect FSH binding, was examined. As with the D404-substitution mutants, rFSHR(t637) was stably expressed but sensitive to Endo H. Significantly, detergent-soluble extracts of cells expressing rFSHR(t637) were unable to bind FSH. From these results, we conclude that substitution of D404 of the rFSHR prevents hormone binding as a result of the intracellular retention of the mutants in the endoplasmic reticulum presumably in an incompletely folded state, as opposed to disruption of a hormone-binding site at D404. Comparable rLHR substitution (D397K) and truncation (t616) mutants were constructed and used to transfect 293 cells. For both rLHR(D397K) and rLHR(t616), human CG (hCG) binding to intact cells was not detectable, but high affinity hCG binding was observed in detergent-soluble extracts of the cells. Therefore, the rLHR differs from the rFSHR in that mutants of the rLHR that are retained in the endoplasmic reticulum have already been folded correctly and can bind hCG with high affinity as long as a hormone-binding site has not been perturbed by the mutation. In contrast, mutants of the rFSHR that are retained in the endoplasmic reticulum have not yet folded into a conformation that can bind hormone. This suggests a difference in the temporal pattern of folding between the two structurally related gonadotropin receptors. Our studies also demonstrate how mutagenesis studies of the rFSHR must be interpreted with caution, as FSHR mutants that are expressed but are retained intracellularly will most likely not be able to bind FSH even when a hormone-binding site has not been altered.
Mol Endocrinol 1995 Dec
PMID:Intracellular retention of mutant gonadotropin receptors results in loss of hormone binding activity of the follitropin receptor but not of the lutropin/choriogonadotropin receptor. 861 9

We have used pulse-chase immunoprecipitations methods to study early post-translational processing of CBI-gp, a lysosomal membrane glycoprotein expressed by African trypanosomes, Rap67, a polyclonal antibody to CBI-gp, immunoprecipitated a 100-kDa glycoprotein, gp100, from both bloodstream forms (BF) and procyclic forms (PF) of Trypanosoma brucei gambiense immediately after a 5-min pulse with radiomethionine. N-Glycanase digestion released a 67-kDa core protein, p67, from gp100 of both life cycle forms V8 protease digestion of p67 from BF and PF yielded 13 identical methionyl peptides, suggesting that gp100 from both life cycle forms have very similar or identical p67 core molecules. In BF, gp 100 carried both endoglycosidase H (EndoH)-resistant and EndoH-sensitive, N-linked oligosaccharides immediately after labeling. In PF, all the N-linked sugars on gp100 were EndoH sensitive. In BF, gp100 chased progressively into slower migrating 150-180-kDa components that obtained the CBI epitope, traveled to the cell surface where they could be biotinylated, and were proteolytically processed. The increase in mass of gp100 during chase in BF resulted from an elongation of N-linked oligosaccharides. Maturation of gp100 into 150-180-kDa CBI-gp was inhibited if BF were chased in the presence of glucosidase inhibitors castanospermine or deoxynojirimycin. In PF, gp100 did not increase in mass, could not be biotinylated on the cell surface, and was not proetolyzed during extended chases. Cryoimmunoelectron microscopy revealed that the antigens detected by rap67 are abundant in lysosomes and endosomes in both BF and PF. Thus, BF and PF express very similar or identical lysosomal membrane glycoproteins but process and transport them in very different ways.
Mol Biochem Parasitol 1995 Nov
PMID:Processing and transport of a lysosomal membrane glycoprotein is developmentally regulated in African trypanosomes. 871 58

The three-dimensional structure of hevamine, a plant enzyme with chitinase and lysozyme activity, has been refined at 1.8 A resolution to an R-factor of 14.9% and a free R-factor of 19.6%. The final model consists of all 273 amino acid residues and 206 ordered water molecules. Two non-proline cis-peptides were identified, involving Phe32 and Trp255, both of which are implicated in substrate binding. Other glycosyl hydrolase family 18 proteins with known three-dimensional structure are bacterial chitinase A, endo-beta-N-acetylglucosaminidase F1, endo-beta-N-acetylglucosaminidase H, and the two plant proteins concanavalin B and narbonin, which have no known enzymatic activity. All these structures contain a (beta alpha)8 barrel fold, with the two family 18 consensus regions roughly corresponding to the third and fourth barrel strands. This confirms the grouping of these proteins into family 18, which was only based on weak and local sequence similarity. The substrate specificity of the enzymes is determined by the loops following the barrel strands that form the substrate binding site. All enzymes have an aspartic acid and a glutamic acid residue in positions identical with Asp 125 and the catalytic Glu127 of hevamine. The lack of chitinase activity of concanavalin B and narbonin can be explained by the absence of one of these carboxylate groups, and by differences in the loops that form the substrate-binding cleft in hevamine.
J Mol Biol 1996 Sep 20
PMID:The 1.8 A resolution structure of hevamine, a plant chitinase/lysozyme, and analysis of the conserved sequence and structure motifs of glycosyl hydrolase family 18. 883 91


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