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)

Lymphocytes were treated with hydrolytic enzymes primarily to assess whether such modified cells would give improved cytotoxicity reactions during tissue typing. Papain-treated and alpha-chymotrypsin-treated lymphocytes were approximately twice as sensitive as untreated cells in the microcytotoxicity test used, and this finding might be usefully exploited by immunological laboratories for purposes of cross-matching, HLA antibody screening and HLA-DR typing. Trypsin treatment promoted massive cell clumping, while neuraminidase treatment was responsible for indiscriminate cell death after exposure to rabbit serum. The capacity of lymphocytes to form rosettes with sheep erythrocytes was abolished after treatment with trypsin or alpha-chymotrypsin, but enhanced by papain or neuraminidase.
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PMID:Enzymatic modification of the lymphocyte surface. Application to tissue typing and rosetting with sheep erythrocytes. 657 19

The effects of the inhibitor of N-linked glycosylation, tunicamycin, on the synthesis of HLA-A and -B antigens in the human lymphoblastoid cell line JY are described. HLA-A and -B antigens are membrane glycoproteins that consist of a two chain complex, the heavy chain being glycosylated at Asn 86, whereas the light chain, identical to beta 2-microglobulin, is not glycosylated. HLA-A and -B antigens synthesized in the presence of the antibiotic are devoid of carbohydrate. This lack of carbohydrate does not affect the association of the heavy and light chains, nor does it affect the reactivity with human alloantisera, or a mouse monoclonal antibody W6/32, that reacts with all HLA-A and -B specificities examined so far. Nonglycosylated HLA-A and -B antigens are no more susceptible to proteolysis with trypsin, chymotrypsin, or papain than their fully glycosylated counterparts. Thus it may be concluded that the carbohydrate side chains of HLA-A and -B antigens do not contribute significantly to the conformation of HLA-A and -B antigens, at least as measured by these procedures. Pulse-chase experiments, in conjunction with the isolation of cell-surface HLA-A and -B antigens by adsorbing the monoclonal antibody W6/32 to intact cells, indicate that nonglycosylated molecules reach the cell surface at a rate indistinguishable from that of fully glycosylated molecules (although the absolute amount synthesized in the presence of tunicamycin was decreased). Thus glycosylation is also not required for membrane insertion of HLA antigens, nor for their subsequent transport to the cell surface.
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PMID:Biosynthesis and cell surface localization of nonglycosylated human histocompatibility antigens. 700 35

The primary structure of the COOH-terminal cyanogen bromide (CNBr) cleavage fragment Ic (CN-Ic) of the extracellular portion of the murine histocompatibility antigen H-2Kb has been completed. CN-Ic contains a site of papain cleavage which has been utilized for solubilizing H-2Kb by cleaving off the membrane integrating portion of the molecule. The amino acid sequence of CN-Ic has been determined by using peptides recovered after trypsin digestion of CN-Ic before and after blockage of lysine groups with citraconic anhydride. Overlapping sequences for the tryptic fragments were obtained by amino-terminal sequence analysis. The sequence of fragment CN-Ic, which spans residues 229-284 in H-2Kb, is as follows: Glu-Leu-Val-Glu-Thr-Arg-Pro-Ala-Gly-Asp-Gly-Thr-Phe-Gln-Lys-Trp-Ala-Ser-Val-Val-Pro-Leu-Gly-Lys-Glu-Gln-Tyr-Tyr-Thr-Cys-His-Val-Tyr-Gln-Gln-Gly-Leu-Pro-Gln-Pro-Leu-Thr-Leu-Arg-Trp-Asp-Glu-Pro-Pro-Ser-Thr-Val-Ser-Asn-Met. This amino acid sequence determination completes the primary structure of the amino terminal 284 residues of H-2Kb, that portion of this histocompatibility antigen which is external to the cell membrane and which contains antigenic determinants. It was also possible to identify Val-281 as a papain cleavage site within CN-Ic. The completed structure was analyzed solely by radiochemical methods. The structure obtained for H-2Kb is 71% homologous to the reported structure of HLA-B7, a human homologue.
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PMID:Primary structure of murine major histocompatibility complex alloantigens: completion of the sequence of the amino-terminal 284 residues of H-2Kb. 747 Apr 58

Langerhans cells (LC) belong to the dendritic cell lineage and are the principal antigen-presenting cells of squamous epithelia. Short-term cultured LC (cLC) exhibit a marked augmented capacity to stimulate allogeneic T cells and acquire the ability to activate naive T cells, probably in relation to enhanced expression of accessory signals. In this study, we evaluated the expression of B7 costimulatory molecule (CD80) in human freshly isolated (fLC) and cLC at both the protein and mRNA level. Staining of frozen skin sections did not reveal any epidermal dendritic cell reactive with either of two different anti-B7 monoclonal antibodies. fLC in suspension did not exhibit any B7 staining as evaluated by two-color flow-cytometry analysis and immunoelectron microscopy. In contrast, LC that were cultured for 24-72 h displayed strong surface B7 reactivity with a characteristic patchy pattern. Treatment with dispase and trypsin did not reduce B7 staining of cLC. Following warming to 37 degrees C, cLC tagged with anti-B7 monoclonal antibody and gold-conjugated secondary antibody could internalize surface B7 by using the organelles of receptor-mediated endocytosis. B7 mRNA, detected by the reverse-transcriptase polymerase chain reaction technique, was expressed at a low level in purified (> 90% HLA-DR+) fLC but not in LC-depleted epidermal cells, and was markedly upregulated in purified cLC. The results indicate that 1) fLC do not express B7 protein on their surface, but acquire B7 during culture, 2) surface B7 is not sensitive to trypsin, 3) B7 expression is regulated primarily at the mRNA level, and 4) membrane B7 can be internalized within cLC. B7 molecule on CLC may be relevant to their increased antigen-presenting cell potency and ability to stimulate naive T lymphocytes.
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PMID:Expression of B7 costimulatory molecule in cultured human epidermal Langerhans cells is regulated at the mRNA level. 751 82

Birch Pollen is one of common causal agents of nasal allergy (pollinosis) in Hokkaido, Japan. We have previously reported the positive association of the pollinosis with HLA-DR9 antigens. It was reported by others that Bet vI, a 17kDa protein with known amino acid sequences represented the major allergen of birch pollen in Europe. We have tried to determine the location of possible allergenic activity on the 17kDa protein of Japanese birch pollen (Betura Platyphylla var. Japonica). In this study, we have examined the lymphocytes proliferative response (LPR) for trypsin digested materials of a 17kDa protein obtained from Betura Platyphylla var. Japonica. The strong response of lymphocyte was observed against the peptide fragments composed of amino acids from 22nd to 33rd (22-33) and from 56th to 66th (56-66) of the Bet vI molecule. It was also found that our 17kDa protein had isoleusine substitution for phenylalanine at position 31. By using synthetic peptides, we showed that asparagine at position 29 of 22-33 and glutamic acid at position 61 of 56-66 were critical for LPR.
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PMID:[Analysis of birch pollen allergen]. 786 55

We have described hereditary incomplete deficiency of the fourth component of complement (C4) in 10 members of a large kindred. C4 deficiency in this kindred is not linked to C4 loci in the HLA region. C4 synthesis is decreased, and C4 catabolism is normal in kindred members with low serum C4 levels. We have discovered a uniquely dysfunctional C1 inhibitor in all C4-deficient members of this kindred. C1 inhibitor dysfunction is revealed by incubating sera of affected members with EDTA, which destroys all C4 activity in these sera, but not in normal sera or sera from individuals with partial C4 deficiencies. The M(r) of C1 inhibitor purified from affected members is normal, but approximately 50% of this C1 inhibitor resists cleavage by trypsin (0.14 microM) at arg444, suggesting a substitution at this position. Moderate increases in trypsin, however, result in cleavage of the resistant molecules, which would not be expected if arg444 were the site of the mutation. All molecules in C1 inhibitor purified from affected members' plasma bind to activated C1s (C1-s), but approximately 50% of molecules in these preparations do not bind to activated C1r (C1r). These findings show that affected kindred members have a unique mutation in C1 inhibitor. The mutant C1 inhibitor does not prevent the activation of C1s by C1-r when serum Ca2+ is chelated by EDTA, but its inhibition of C1-s is normal in vivo, as shown by normal C2 levels, normal C4 catabolism, and absence of angioedema in C4-deficient members. The nature of the mutation, its selective failure to inhibit C1-r, and its relationship to decreased C4 synthesis remain to be defined.
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PMID:Unique C1 inhibitor dysfunction in a kindred without angioedema. I. A mutant C1 INH that inhibits C1-s but not C1-r. 814 14

Regarding their endocrine and paracrine activities, endothelins can be considered as peptide hormones and growth factors. The presence of endothelin-1 (ET-1)-binding sites on smooth muscle of placental villous vessels, on villous trophoblast and on purified trophoblast in culture raises the question of the origin of the peptide. In placenta, endothelin could derive from maternal, fetal and/or endogenous sources. Therefore, localization of ET-1 was investigated by use of immunohistochemistry in human term placenta and in cultured trophoblast using the avidin-biotin-peroxidase complex procedure. Specificity of immunostaining was demonstrated by applying ET-1 antibody that has been preabsorbed with excess peptide. In sections of placenta, ET-1 immunoreactivity (ET-1 IR) was specifically detected in the endothelium of the vessels and in the syncytiotrophoblast of the villi. ET-1 IR was also detected in the decidua-like cells and in the extravillous cytotrophoblast of the basal plate, a region where the maternal and fetal cells intermingle closely. The extravillous cytotrophoblast of the chorionic plate and of the placental septa also exhibited a strong ET-1 IR. For trophoblast culture cytotrophoblastic cells were obtained from placental villi by trypsin-DNase dispersion, further purified on Percoll gradient and enriched by employing a monoclonal anti-HLA class-I antibody. The trophoblastic origin of the cells was demonstrated by immunohistochemistry and by studying the secretion of gestational hormones during culture. After different periods of culture of purified cytotrophoblastic cells (1 to 5 days), ET-1 IR was observed in 95% of cells: cytotrophoblastic cells, trophoblast aggregates, and syncytiotrophoblast.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Localization and production of immunoreactive endothelin-1 in the trophoblast of human placenta. 847 6

Mycobacterium leprae heat-shock proteins hsp65 and hsp18 have received immense attention as major T-cell target antigens in leprosy. Both of these hsps and their tryptic fragments were characterized for their ability to stimulate CD4+ T cells derived from polar leprosy cases and healthy contacts. The optimal digestion of hsps with trypsin yielded four fragments of hsp65--TDB65-1 (24 kDa), TDB65-2 (18 kDa), TDB65-3 (17 kDa), TDB65-4 (14 kDa)-- and three of hsp18--TDB18-1 (10 kDa), TDB18-2 (5 kDa), TDB18-3 (3 kDa). While all of these tryptic fragments and undigested hsps triggered CD4+ T cells from tuberculoid (TT) leprosy patients and healthy contacts (SI > 2), only two fragments--TDB65-2 and TDB18-3--were found to be stimulatory in anergic lepromatous (LL) leprosy patients (SI = 5.27 and 3.0, respectively). Blocking studies using allele-specific anti-DR monoclonal antibodies revealed multiple HLA-Dr restriction, with DR2 providing the strongest restriction in both TT as well as LL leprosy. These findings indicate that M. leprae hsps and their trypsin-digested fragments are promiscuous and recognizable in the context of diverse HLA alleles, of which DR2 is the most efficient restriction element. The 18-kDa fragment of hsp65 and the 3-kDa fragment of hsp18 are the most versatile fragments that could elicit in vitro proliferation in both polar forms of leprosy.
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PMID:CD4+ T-cell responses to recombinant hsp65 and hsp18 of M. leprae and their trypsin-digested fragments in leprosy: diversity in HLA-DR restriction. 864 14

Allergy to birch pollen is common in the Northern Europe, North America, and in Japan, mainly in Hokkaido Island. Previously, we have reported the positive association of birch pollen allergy in HLA-DR9 antigen. We also have identified the recognition sites in 17kDa protein (Bet v1), known as the major allergen of birch pollen, by using T cell proliferation assay against the trypsin digested materials of the 17kDa protein. In this study, overlapping synthetic peptides correspond to the Bet v1 sequences were used to investigate the specificity of T cell responses in two HLA-DR9 positive patients. Three of the epitopes, residues 35-52, 75-92, and 102-119, were recognized by T cells from both patients. These three epitopes include HLA-DR9 binding motifs. Then we established T cell clone specific to p75-92 residue. 78-89 peptide was the core sequence of the epitope for the T cell clone contained the anchor residues characteristic to HLA-DR9. Within the core sequence, five residues were identified as critical for recognition on the basis of inhibition of the T cell proliferative response against singly substituted analogue peptides.
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PMID:[Analysis of T cell epitopes on birch pollen allergen]. 908 63

The release of cytotoxic granule contents by cytotoxic T lymphocytes triggers apoptotic target cell death. Cytotoxic granules contain a pore-forming protein, perforin, and a group of serine proteases called granzymes. We expressed human granzyme A in bacteria as a proenzyme capable of in vitro activation by enterokinase. The recombinant activated enzyme has catalytic activity against substrates with Arg, preferably, or Lys at the P1 position, comparable to trypsin. An enzymatically inactive recombinant granzyme A, with the active site Ser mutated to Ala, was produced and used with affinity chromatography to identify potential substrates. Two granzyme A-binding cytoplasmic proteins of molecular mass 33 and 44 kDa were isolated and identified by tryptic fragment sequencing as PHAP I and II, ubiquitous putative HLA-associated proteins, previously coisolated by binding to an HLA class II peptide. PHAP II forms an SDS-stable complex with recombinant mutant granzyme A and coprecipitates with it from cytoplasmic extracts. PHAP II, either purified or in cell lysates, is cleaved by the recombinant enzyme at nanomolar concentrations to a 25-kDa fragment. PHAP II begins to be degraded within minutes of initiation of cytotoxic T lymphocyte attack. PHAP I and II are candidate participants in the granzyme A pathway of cell-mediated cytotoxicity.
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PMID:Recombinant human granzyme A binds to two putative HLA-associated proteins and cleaves one of them. 925 74


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