Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P20645 (mannose-6-phosphate receptor)
 320 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A monoclonal antibody (2C5) raised against rat liver lysosomal membranes was used to identify a 78-kD glycoprotein that is present in the membranes of both endosomes and lysosomes and, therefore, is designated endolyn-78. In cultures of rat hepatoma (Fu5C8) and kidney cells (NRK), this glycoprotein could not be labeled with [35S]methionine or with [32P]inorganic phosphate but was easily labeled with [35S]cysteine and [3H]mannose. Pulse-chase experiments and determinations of endoglycosidase H (endo H) sensitivity showed that endolyn-78 is derived from a precursor of Mr 58-62 kD that is processed to the mature form with a t1/2 of 15-30 min. The protein has a 22-kD polypeptide backbone that is detected after a brief pulse in tunicamycin-treated cells. During a chase in the presence of the drug, this is converted into an O-glycosylated product of 46 kD that despite the absence of N-linked oligosaccharides is effectively transferred to lysosomes. This demonstrates that the delivery of endolyn-78 to this organelle is not mediated by the mannose-6-phosphate receptor (MPR). Immunocytochemical experiments showed that endolyn-78 is present in the limiting membranes and the interior membranous structures of morphologically identifiable secondary lysosomes that contain the lysosomal hydrolase beta-glucuronidase, lack the MPR, and could not be labeled with alpha-2-macroglobulin at 18.5 degrees C, a temperature which prevents appearance of endocytosed markers in lysosomes. Endolyn-78 was present at low levels in the plasma membrane and in peripheral tubular endosomes, but was prominent in morphologically diverse components of the endosomal compartment (vacuolar endosomes and various types of multivesicular bodies) which acquired alpha-2-macroglobulin at 18.5 degrees C, and frequently contained substantial levels of the MPR and variable levels of beta-glucuronidase. On the other hand, the MPR was very rarely found in endolyn-containing structures that were not labeled with alpha-2-macroglobulin at the low temperature. Thus, the process of lysosomal maturation appears to involve the progressive delivery of lysosomal enzymes to various types of endosomes that may have already received some of the lysosomal membrane proteins. Although endolyn-78 would be one of the proteins added early to endosomes, other lysosomal membrane proteins may be added only to multivesicular endosomes that represent very advanced stages of maturation.
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PMID:Endolyn-78, a membrane glycoprotein present in morphologically diverse components of the endosomal and lysosomal compartments: implications for lysosome biogenesis. 265 37

Menkes disease is a fatal neurodegenerative disorder of childhood due to the absence or dysfunction of a putative copper-transporting P-type ATPase encoded on the X chromosome. To elucidate the biosynthesis and subcellular localization of this protein, polyclonal antisera were generated against a bacterial fusion protein encoding the 4th to 6th copper-binding domains in the amino terminus of the human Menkes protein. RNA blot analysis revealed abundant Menkes gene expression in several cell lines, and immunoblotting studies utilizing this antiserum readily detected a 178-kDa protein in lysates from these cells. Pulse-chase studies indicate that this protein is synthesized as a single-chain polypeptide which is modified by N-linked glycosylation to a mature endoglycosidase H-resistant form. Sucrose gradient fractionation of HeLa cell lysates followed by immunoblotting of individual fractions with antibodies to proteins of known intracellular location identified the Menkes ATPase in fractions similar to those containing the cation-independent mannose-6-phosphate receptor. Consistent with this observation, confocal immunofluorescence studies of these same cells localized this protein to the trans-Golgi network and a vesicular compartment with no expression in the nucleus or on the plasma membrane. Taken together, these data provide a unique model of copper transport into the secretory pathway of mammalian cells which is compatible with clinical observations in affected patients and with recent data on homologous proteins identified in prokaryotes and yeast.
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PMID:Biochemical characterization and intracellular localization of the Menkes disease protein. 894 55

The lysosomal storage disorders are a group of inherited metabolic diseases each characterised by a relative or absolute deficiency of one or more of the lysosomal proteins involved in the hydrolysis of glycoconjugates or in the transport of the resulting product. Enzyme replacement therapies are under consideration for a number of these disorders and are based on the in vitro observation that cells from affected patients can be corrected by addition of exogenous enzyme. In this study, two glycosylation variants of the lysosomal enzyme N-acetylgalactosamine-4-sulphatase (4S) (the deficiency of which causes Mucopolysaccharidosis (MPS) type VI, (Maroteaux-Lamy syndrome) were made by expression of 4S cDNA in both wild type chinese hamster ovary (CHO-K1), and Lec1 (N-acetylglucosaminyltransferase I deficient CHO-K1) cells. Differences in the glycosylation pattern of the two enzyme forms were demonstrated with endoglycosidase H and N-glycosidase F digestions. The receptor mediated binding of these two forms of 4S to two cell types, human skin fibroblasts and rat alveolar macrophages, was then analysed. We have shown that both enzyme forms bind to the mannose-6-phosphate receptor on human skin fibroblasts with equal affinity demonstrating that the degree of phosphorylation of mannose residues in the two forms is similar. However, using rat alveolar macrophages, we found that the binding/uptake of the two enzymes differs considerably. These results show that differences in glycosylation of lysosomal enzymes can be an important factor in altering enzyme uptake by different cell types. Thus, producing carbohydrate modification variants in this way may be useful for altering the distribution of exogenous enzyme in vivo.
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PMID:Receptor mediated binding of two glycosylation forms of N-acetylgalactosamine-4-sulphatase. 963 Jun 76

GD3 synthase (Sial-T2) is a key enzyme of ganglioside synthesis that, in concert with GM2 synthase (GalNAc-T), regulates the ratio of a- and b-pathway gangliosides. In this work, we study the sub-Golgi location of an epitope-tagged version of chicken Sial-T2 transfected to CHO-K1 cells. The expressed protein was enzymatically active both in vitro and in vivo and showed a molecular mass of approximately 47 or approximately 95 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the presence or absence of, respectively, beta-mercaptoethanol. The 95-kDa form of Sial-T2 was also detected if the protein was retained in the endoplasmic reticulum (ER) due to impaired glycosylation, indicating that it was formed in the ER. Confocal immunofluorescence microscopy showed Sial-T2 localized to the Golgi complex and, within the organelle, partially co-localizing with the mannose-6-phosphate receptor, a marker of the trans-Golgi network (TGN). In cells treated with brefeldin A, a major fraction of Sial-T2 redistributed to the ER, even under controlled expression to control for mislocalization due to protein overloading. In experiments of incorporation of sugars into endogenous acceptors of Golgi membranes in vitro, GD3 molecules formed by incubation with CMP-NeuAc were converted to GD2 upon incubation with UDP-GalNAc. These results indicate that Sial-T2 localizes mainly to the proximal Golgi, although a fraction is located in the TGN functionally coupled to GalNAc-T. Consistent with this, most of the enzyme was in an endoglycosidase H (Endo-H)-sensitive, neuraminidase (NANase)-insensitive form. A minor secreted form lacking approximately 40 amino acids was Endo-H-resistant and NANase-sensitive, indicating that the cells were able to process N-glycans to Endo-H-resistant forms. Taken together, the results of these biochemical and immunocytochemical experiments indicate that in CHO-K1 cells, most Sial-T2 localizes in the proximal Golgi and that a functional fraction is also present in the TGN.
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PMID:GM3 alpha2,8-sialyltransferase (GD3 synthase): protein characterization and sub-golgi location in CHO-K1 cells. 1073 30

XTP3-B is a soluble endoplasmic reticulum (ER)-resident protein containing two mannose-6-phosphate receptor homology (MRH) domains in its sequence. XTP3-B interacts with a membrane-associated ubiquitin ligase complex, and, therefore, is thought to participate in ER-associated degradation (ERAD). In this study, the recombinant human XTP3-B fused with IgG-Fc (XTP3-B-Fc), XTP3-B without an N-terminal MRH domain fused with IgG-Fc (XTP3-BDelta1-Fc), or XTP3-B without a C-terminal MRH domain fused with IgG-Fc (XTP3-BDelta2-Fc) were prepared. XTP3-B-Fc and XTP3-BDelta1-Fc bound to Lec1 cells but not to CHO, Lec2, or Lec8 cells, while XTP3-BDelta2-Fc did not bind to any of these cells. The binding of XTP3-B-Fc and XTP3-BDelta1-Fc to Lec1 cells was abrogated by treatment of the cells with endo-beta-N-acetylglucosaminidase H, Manalpha1,6Man or Manalpha1,6(Manalpha1,3)Manalpha1,6(Manalpha1,3)Man, or by substitution of Arg428 or Tyr457 in the C-terminal MRH domain with alanine. Arg428 and Tyr457 are homologous to amino acids that mediate glycan binding by the cation-dependent mannose-6-phosphate receptor. An immunoprecipitation experiment using lysates of cells co-expressing wild-type alpha1-antitrypsin (AT), alpha1-antitrypsin variant null(Hong Kong) (AT(NHK)), and FLAG-tagged XTP3-B, or its mutants, demonstrated that AT(NHK), but not AT, specifically co-precipitated with XTP3-B and XTP3-BDelta1. The glycan-binding-deficient XTP3-BDelta2 did not bind either AT or AT(NHK). These results suggest that XTP3-B specifically binds to AT(NHK), which is a well-known substrate of ERAD, via a C-terminal MRH domain in a glycan-dependent manner.
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PMID:Human XTP3-B binds to alpha1-antitrypsin variant null(Hong Kong) via the C-terminal MRH domain in a glycan-dependent manner. 1991 67