Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.4.21.4 (trypsin)
42,187 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Many neuroendocrine precursor proteins, such as proopiomelanocortin (POMC), are cleaved in a tissue specific manner at distinct pairs of basic amino acids. Elucidating the specificity of the prohormone endoprotease(s) is essential to understanding cleavage specificity. However, isolation of these enzymes has been difficult, due to the inability to distinguish authentic maturation enzyme from the many other trypsin-like activities present in tissue homogenates. Recently, a "signature" of the insulin cell endoprotease(s) was defined in vivo by assessing the processing of a series of mutant cleavage sites in a model prohormone, mouse POMC (mPOMC) (Thorne, B. A., and Thomas, G. (1990) J. Biol. Chem. 265, 8436-8443. To investigate mechanisms of tissue-specific processing, we sought to identify the endoprotease signature of a cell having a processing phenotype distinct from insulinoma cells. In this report, the cleavage site specificity of the endoprotease(s) expressed in bovine adrenal chromaffin cells is examined. High levels of mPOMC (1.6 pmol/10(6) cells) were expressed in these cells using a vaccinia virus vector, and the precursor was targeted to the regulated secretory pathway. Analysis of POMC-derived peptides revealed that chromaffin cells processed the prohormone to a set of peptides highly similar to anterior pituitary corticotrophs, including adrenocorticotropin hormone (ACTH) and beta-lipotropin, gamma-lipotropin, and beta-endorphin. This processing contrasted with the pattern of cleavage site utilization in Rin m5F insulinoma cells, which more closely resembled that of the intermediate pituitary melanotrophs. However, the processing preference for the sequences of pairs of basic amino acids (as tested using the entire series of mutant cleavage sites; -LysArg- (native), -ArgArg-, -ArgLys-, -LysLys-, -HisArg-, -MetArg- at the ACTH/beta-lipotropin junction and -LysLys- (native), -LysArg-, -ArgArg-, -ArgLys- in beta-endorphin) was the same in both insulinoma and adrenal chromaffin cells, suggesting recognition and cleavage by similar enzymes in both cell types. The cell-specific processing of mPOMC may thus result from expression of a common core set of processing enzymes and factors unique to each cell type affecting the enzyme accessibility to precursor cleavage sites.
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PMID:Expression and processing of mouse proopiomelanocortin in bovine adrenal chromaffin cells. A model system to study tissue-specific prohormone processing. 185 97

A protein with pancreastatin-like immunoreactivity has been isolated and purified from liver metastasis of a patient with insulinoma. NH2-terminal residue analysis, in conjunction with the use of antibodies that are specific for the C-terminal amide peptide of porcine pancreastatin, identified this protein as a 186-amino-acid protein corresponding to human chromogranin A-116-301 (the fragment corresponding to the positions from 116 to 301 of human chromogranin A). Digestion of this protein with trypsin yielded a 48-amino-acid peptide with the retention of full pancreastatin activity. Serum from patient with insulinoma contains a peptide specie(s) that comigrates with the 48-amino-acid pancreastatin, suggesting that this peptide might be a physiologically important circulation form of pancreastatin in humans. A sensitive radioimmunoassay was established using antibody developed against a synthetic 29-amino-acid peptide amide of pancreastatin. Immunocytochemical staining revealed that a major population of human pancreatic islet cells were immunoreactive to the antiserum but with varying intensity of staining. Pancreastatin-like immunoreactivity was not observed in exocrine acinar cells.
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PMID:Isolation and characterization of a tumor-derived human protein related to chromogranin A and its in vitro conversion to human pancreastatin-48. 216 9

Monoclonal antibody (Mab) 1.93B7 was obtained by fusion of spleen cells from a diabetic NOD mouse with P3X63Ag8.653 myeloma cells and screening for complement mediated lysis of rat insulinoma (RIN) cells. Immunofluorescence studies revealed that this Mab binds to RIN cells but not to the rat pituitary tumour line GH3. The binding of Mab 1.93B7 to RIN cells was abolished by trypsin but not by neuraminidase treatment of the cells, suggesting that the antigen recognized is a protein. Mab 1.93B7 bound to approximately 30% of mouse (BALB/c) and rat islet cells which had been subjected to trypsin digestion and incubated as a single cell suspension for 12h to allow reexpression of trypsin sensitive antigens. Since Mab 1.93B7 is potentially pathogenic, as suggested by its reactivity to primary islet cells and its complement fixing capacity, we injected it into BALB/c and NOD mice. Cytotoxic activity against RIN cells was detected in the serum of the animals injected with Mab 1.93B7, but the Mab did not exert a diabetogenic action and failed to reverse diabetes when administered at onset in NOD mice. No modification of the course of spleen cell mediated transfer of diabetes in NOD mice was observed when the Mab was administered from the time of spleen cell inoculation to the appearance of glycosuria. The implications of the lack of an effect in vivo of Mab 1.93B7 under the conditions employed are discussed.
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PMID:A cytotoxic monoclonal islet cell surface antibody from the NOD mouse. 269 57

Monoclonal antibody 3A4 to islet cell surface antigen has been previously established in our laboratory, using hybridization of spleen lymphocytes from non-obese diabetic (NOD) mice transferred into immunologically incompetent recipient mice. In the present study, monoclonal islet cell surface antibody 5C12 could be newly obtained in the 10:1 ratio of NOD mice spleen cells and mouse myeloma cells (SP2/0) without any modifications. Protein A radioligand assay and indirect immunofluorescence on living cells showed that 5C12 antibody reacted to normal rat islet cells and cultured rat insulinoma cells (RIN-r), but not to cultured lymphocytes (Bri-7, IM-9) and Chang-liver cells. Analysis of 125I-labeled antibody binding revealed that unlabeled 5C12 effectively inhibited subsequent 125I-5C12 binding to RIN-r cells, whereas unlabeled 3A4 did not. The scatchard plot from these data showed the curvilinearity, and about 150,000 binding sites to antibody per RIN-r cell were counted. The treatment of RIN-r cells with papain and neuraminidase reduced the binding of 5C12 to RIN-r cells, whereas the effect of trypsin was not observed. Immunoprecipitation of 125I-labeled insulinoma cell lysates followed by SDS-PAGE and autoradiography indicated that 5C12 recognized 105K dalton cell surface protein in RIN-r cells. Immunoblotting also showed that 5C12 antibody recognized 105K dalton cell surface protein in RIN-r cells. These results demonstrated that 5C12 was an important tool for clarifying the immunoresponse against certain antigenic determinants on pancreatic B cells. Furthermore, 5C12 has not only qualitatively and quantitatively improved diagnostic methodology, but it may also provide new reagents useful to the treatment and prevention of type 1 diabetes.
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PMID:[An analysis of islet cell surface antigen defined by monoclonal islet cell surface antibody 5C12]. 354 94

An islet cell tumor, characterized by proinsulin level significantly elevated above normal human pancreas, has been found to contain insulin- and glucagon-degrading activity. Examination by chromatography on Sephadex G-75 of the degradation products formed from insulin showed A chain, and B chain rich-A chain aggregate as previously found with rat pancreatic islets. There was, however, little conversion of A chain to low molecular weight components indicating that insulinoma peptidase that has been found to degrade glucagon at about pH 6.8 degraded that A chain to a markedly lower rate. In contrast to the insulin-degrading activity, which was activated by glutathione in the presence of EDTA, the peptidase activity was not affected by the thiol compound. The activity of the peptidase was markedly inhibited by chelating agents, i.e., EDTA and o-phenanthroline, whereas chymotrypsin and trypsin inhibitors, i.e., TOS-PheCH2Cl, TOS-LysCH2Cl, soybean and pancreas trypsin inhibitor were found to have no effect.
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PMID:Thiol-protein disulfide oxidoreductase and peptidase activities in insulinoma tissue. 632 44

Major targets for autoantibodies associated with the development of insulin-dependent diabetes mellitus (IDDM) include tryptic fragments with a molecular mass of 37 kDa and/or 40 kDa of a pancreatic islet cell antigen of unknown identity. The assay identifying autoantibodies against the 37/40-kDa antigen in human sera is based on the immunoprecipitation of 35S-labeled rat insulinoma cell proteins with sera from IDDM patients, followed by limited trypsin digestion of the immunoprecipitated material. To identify cDNA clones coding for the 37/40-kDa antigen, we have screened a cDNA expression library from rat insulinoma cells with a serum from an IDDM patient that precipitated the 37/40-kDa antigen in our assay. Among the cDNA products that reacted with the IDDM serum, we identified one cDNA clone whose open reading frame encodes a protein with a predicted mass of 105 kDa that we termed "ICA105" for 105-kDa islet cell antibody. The deduced amino acid sequence has high homology to a recently cloned putative tyrosine phosphatase IA-2 from human and mouse cDNA libraries. Translation of the cDNA in vitro results in a polypeptide with the expected molecular mass of 105 kDa. The evidence that ICA105 is indeed the precursor of the 37/40-kDa tryptic fragments is based on the following three results: (i) Sera from IDDM patients containing autoantibodies to the 37/40-kDa antigen precipitate the in vitro translated polypeptide, whereas sera from healthy subjects as well as sera from IDDM patients not reactive with the 37/40-kDa antigen do not precipitate the cDNA product. (ii) Immunoprecipitation of the in vitro translated protein with sera containing autoantibodies to the 37/40-kDa antigen followed by limited trypsin digestion of the precipitated proteins results in a 40-kDa polypeptide. (iii) The protein derived from our cDNA but not from an unrelated control cDNA clone can block immunoprecipitation of the 37/40-kDa antigen from a labeled rat insulinoma cell extract. The availability of the cloned 37/40-kDa antigen should facilitate the identification of individuals at risk of IDDM with increased accuracy. Furthermore, the identification of the 37/40-kDa antigen as the putative tyrosine phosphatase IA-2 is of relevance in elucidating the role of this antigen in the development of IDDM.
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PMID:The 37/40-kilodalton autoantigen in insulin-dependent diabetes mellitus is the putative tyrosine phosphatase IA-2. 756 43

Sera from patients with insulin-dependent diabetes immunoprecipitate 64,000-M(r) proteins, distinct from glutamate decarboxylase, that are cleaved to 37,000- and 40,000-M(r) fragments by trypsin. We investigated possible relationships between 37,000- or 40,000-M(r) fragments of antigen and the tyrosine phosphatase-like protein, IA-2 (ICA512). Antibodies from nondiabetic relatives bound differentially to 37,000- and 40,000-M(r) fragments indicating presence of distinct epitopes. Precursors of these fragments could be separated on immobilized lectins, suggesting different carbohydrate content. Levels of antibodies to 40,000-M(r) fragments were strongly associated with those to the intracellular domain of IA-2. Recombinant intracellular domain of IA-2 blocked binding of antibodies to 40,000-M(r) fragments expressed by insulinoma cells and partially blocked binding to 37,000-M(r) fragments. Furthermore, trypsinization of recombinant intracellular domain of IA-2 generated proteolytic fragments of identical M(r) to the 40,000-M(r) fragments of insulinoma antigen; 37,000-M(r) fragments were not generated. Thus, 40,000-M(r) fragments of islet autoantigen are derived from a protein similar or identical to the tyrosine phosphatase-like molecule, IA-2. The 37,000-M(r) fragments are derived from a different, although related, protein.
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PMID:Relationship of the 37,000- and 40,000-M(r) tryptic fragments of islet antigens in insulin-dependent diabetes to the protein tyrosine phosphatase-like molecule IA-2 (ICA512). 765 22

The biosynthesis, intracellular transport, and surface expression of the beta cell glucose transporter GLUT2 was investigated in isolated islets and insulinoma cells. Using a trypsin sensitivity assay to measure cell surface expression, we determined that: (a) greater than 95% of GLUT2 was expressed on the plasma membrane; (b) GLUT2 did not recycle in intracellular vesicles; and (c) after trypsin treatment, reexpression of the intact transporter occurred with a t1/2 of approximately 7 h. Kinetics of intracellular transport of GLUT2 was investigated in pulse-labeling experiments combined with glycosidase treatment and the trypsin sensitivity assay. We determined that transport from the endoplasmic reticulum to the trans-Golgi network (TGN) occurred with a t1/2 of 15 min and that transport from the TGN to the plasma membrane required a similar half-time. When added at the start of a pulse-labeling experiment, brefeldin A prevented exit of GLUT2 from the endoplasmic reticulum. When the transporter was first accumulated in the TGN during a 15-min period of chase, but not following a low temperature (22 degrees C) incubation, addition of brefeldin A (BFA) prevented subsequent surface expression of the transporter. This indicated that brefeldin A prevented GLUT2 exit from the TGN by acting at a site proximal to the 22 degrees C block. Together, these data demonstrate that GLUT2 surface expression in beta cells is via the constitutive pathway, that transport can be blocked by BFA at two distinct steps and that once on the surface, GLUT2 does not recycle in intracellular vesicles.
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PMID:GLUT2 surface expression and intracellular transport via the constitutive pathway in pancreatic beta cells and insulinoma: evidence for a block in trans-Golgi network exit by brefeldin A. 827 90

Patients with insulin-dependent diabetes (IDDM) possess antibodies to islet proteins of M(r)-64,000. Potential autoantigens of this M(r) include glutamate decarboxylase (GAD) and 65 kD heat shock protein. We have detected two distinct antibody specificities in IDDM that bind 50,000 M(r) or 37,000/40,000 M(r) proteolytic fragments of 64,000 M(r) proteins. In this study, we investigated relationships of these proteolytic fragments to GAD and heat shock proteins. Polyclonal antibodies to GAD bound 50,000 M(r) fragments of islet antigen. Recombinant GAD65, but not GAD67, blocked binding to this antigen, suggesting that 50,000 M(r) fragments are derived from islet GAD65. In contrast, GAD antibodies did not recognize 37,000/40,000 M(r) fragments, and neither GAD isoforms blocked autoantibody binding to precursors of these fragments. The 37,000/40,000 M(r) fragments, but not the 50,000 M(r) fragments, were detected after trypsin treatment of immunoprecipitates from insulinoma cells that lacked expression of major GAD isoforms. Antibodies in IDDM did not bind native or trypsinized islet heat shock proteins. Thus, IDDM patients possess antibodies to GAD, but also distinct antibodies to a 64,000 M(r) protein that is not related to known GAD isoforms or heat shock proteins.
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PMID:Detection of pancreatic islet 64,000 M(r) autoantigens in insulin-dependent diabetes distinct from glutamate decarboxylase. 832 89

A novel cDNA, IA-2beta, was isolated from a mouse neonatal brain library. The predicted protein sequence revealed an extracellular domain, a transmembrane region, and an intracellular domain. The intracellular domain is 376 amino acids long and 74% identical to the intracellular domain of IA-2, a major autoantigen in insulin-dependent diabetes mellitus (IDDM). A partial sequence of the extracellular domain of IA-2beta indicates that it differs substantially (only 26% identical) from that of IA-2. Both molecules are expressed in islets and brain tissue. Forty-six percent (23 of 50) of the IDDM sera but none of the sera from normal controls (0 of 50) immunoprecipitated the intracellular domain of IA-2beta. Competitive inhibition experiments showed that IDDM sera have autoantibodies that recognize both common and distinct determinants on IA-2 and IA-2beta. Many IDDM sera are known to immunoprecipitate 37-kDa and 40-kDa tryptic fragments from islet cells, but the identity of the precursor protein(s) has remained elusive. The current study shows that treatment of recombinant IA-2beta and IA-2 with trypsin yields a 37-kDa fragment and a 40-kDa fragment, respectively, and that these fragments can be immunoprecipitated with diabetic sera. Absorption of diabetic sera with unlabeled recombinant IA-2 or IA-2beta, prior to incubation with radiolabeled 37-kDa and 40-kDa tryptic fragments derived from insulinoma or glucagonoma cells, blocks the immunoprecipitation of both of these radiolabeled tryptic fragments. We conclude that IA-2beta and IA-2 are the precursors of the 37-kDa and 40-kDa islet cell autoantigens, respectively, and that both IA-2 and IA-2beta are major autoantigens in IDDM.
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PMID:Identification of a second transmembrane protein tyrosine phosphatase, IA-2beta, as an autoantigen in insulin-dependent diabetes mellitus: precursor of the 37-kDa tryptic fragment. 863 68


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