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)

We have cloned and characterized a cDNA encoding a new human serine proteinase, testisin, that is abundantly expressed only in the testis and is lost in testicular tumors. The testisin cDNA was identified by homology cloning using degenerate primers directed at conserved sequence motifs within the catalytic regions of serine proteinases. It is 1073 nucleotides long, including 942 nucleotides of open reading frame and a 113-nucleotide 3' untranslated sequence. Northern and dot blot analyses of RNA from a range of normal human tissues revealed a 1.4-kb mRNA species that was present only in testis, which was not detected in eight of eight testicular tumors. Testisin cDNA is predicted to encode a protein of 314 amino acids, which consists of a 19-amino acid (aa) signal peptide, a 22-aa proregion, and a 273-aa catalytic domain, including a unique 17-aa COOH-terminal hydrophobic extension that is predicted to function as a membrane anchor. The deduced amino acid sequence of testisin shows 44% identity to prostasin and contains features that are typical of serine proteinases with trypsin-like substrate specificity. Antipeptide antibodies directed against the testisin polypeptide detected an immunoreactive testisin protein of Mr 35,000-39,000 in cell lysates from COS-7 cells that were transiently transfected with testisin cDNA. Immunostaining of normal testicular tissue showed that testisin was expressed in the cytoplasm and on the plasma membrane of premeiotic germ cells. No staining was detected in eight of eight germ cell-derived testicular tumors. In addition, the testisin gene was localized by fluorescence in situ hybridization to the short arm of human chromosome 16 (16p13.3), a region that has been associated with allellic imbalance and loss of heterozygosity in sporadic testicular tumors. These findings demonstrate a new cell surface serine proteinase, loss of which may have a direct or indirect role in the progression of testicular tumors of germ cell origin.
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PMID:Testisin, a new human serine proteinase expressed by premeiotic testicular germ cells and lost in testicular germ cell tumors. 1039 66

Fusion of the lentogenic strain "Clone 30" of Newcastle disease virus (NDV) with the cell line COS-7 has been studied. Fusion was monitored using the octadecylrhodamine B chloride dequenching assay [Hoekstra, D., de Boer, T., Klappe, K. and Wilschut, J. (1984). Biochemistry 23, 5675-5681]. In the present work, fusion of NDV with COS-7 cells was found to occur in a time- and temperature-dependent fashion. Significant dequenching of the probe occurred at temperatures higher than 28 degrees C. A 20-fold excess of unlabeled virus inhibited fusion by about 53% compared with the control, whereas 62% inhibition of fusion was obtained after digestion of viral glycoproteins with trypsin. The data are discussed in terms of the nonfusion transfer of the probe. In addition, preincubation of cells with 50 mM ammonium chloride or 0.1% sodium azide prevented NDV from fusing with COS-7 cells by about 30% in comparison with the control. The cytopathic effect of NDV infection in cell culture in the presence of ammonium chloride was reduced compared with control. Moreover, viral preincubation at pH 5 yielded a mild inhibition of fusogenic activity. Our results suggest that NDV may use the endocytic pathway as a complementary way of entering cells by direct fusion with the plasma membrane.
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PMID:Acidic pH enhancement of the fusion of Newcastle disease virus with cultured cells. 1041 67

To reexamine the existing predictions about the general membrane topology of the high-affinity Na+/glucose cotransporter (SGLT1) and in particular of the large loop at the C-terminal region, a small 6 x Histidine-tag was introduced at different positions of the SGLT1 sequence by site-directed mutagenesis. Eleven His-SGLT1 mutants were constructed and were transiently transfected into COS-7 cells. As demonstrated by immunofluorescent labeling with antipeptide antibodies against SGLT1, all mutants were expressed and inserted into the plasma membrane. Only mutants with the tag in the N-terminal region and the C-terminal region retained Na+/glucose cotransport activity at 0.1 mM D-glucose. The arrangement of the His-tag in the membrane was analyzed by indirect immunofluorescence, using a monoclonal antihistidine antibody. In nonpermeabilized cells the His-tag could be detected at the N-terminal end (insertion at aa 5) and at the C-terminal end (replacement between aa 584-589 and between aa 622-627), suggesting that these portions of the polypeptide are accessible from the extracellular space. Furthermore, an epitope-specific antibody directed against aa 606-630 reacted strongly with the cell surface. To support this topology intact stably transfected SGLT1 competent CHO cells were partially digested with an immobilized trypsin and subsequently subjected to electrophoresis and Western blot analysis. The size of the digestion product suggests that extravesicular trypsin removed the extracellular loop that contains the amino acid residues 549-664. Thus our results indicate that the last large loop (about aa 541-aa 639) towards the C-terminal end faces the cell exterior where it might be involved in substrate recognition.
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PMID:Probing transmembrane topology of the high-affinity Sodium/Glucose cotransporter (SGLT1) with histidine-tagged mutants. 1044 67

Lactase-phlorizin hydrolase (LPH) is a membrane bound intestinal hydrolase, with an extracellular domain comprising 4 homologous regions. LPH is synthesized as a large polypeptide precursor, pro-LPH, that undergoes several intra- and extracellular proteolytic steps to generate the final brush-border membrane form LPHbeta(final). Pro-LPH is associated through homologous domain IV with the membrane through a transmembrane domain. A truncation of 236 amino acids at the COOH terminus of domain IV (denoted LAC236) does not significantly influence the transport competence of the generated mutant LPH1646MACT (Panzer, P., Preuss, U., Joberty, G., and Naim, H. Y. (1998) J. Biol. Chem. 273, 13861-13869), strongly suggesting that LAC236 is an autonomously folded domain that links the ectodomain with the transmembrane region. Here, we examine this hypothesis by engineering several N-linked glycosylation sites into LAC236. Transient expression of the cDNA constructs in COS-1 cells confirm glycosylation of the introduced sites. The N-glycosyl pro-LPH mutants are transported to the Golgi apparatus at substantially reduced rates as compared with wild-type pro-LPH. Alterations in LAC236 appear to sterically hinder the generation of stable dimeric trypsin-resistant pro-LPH forms. Individual expression of chimeras containing LAC236, the transmembrane domain and cytoplasmic tail of pro-LPH and GFP as a reporter gene (denoted LAC236-GFP) lends strong support to this view: while LAC236-GFP is capable of forming dimers per se, its N-glycosyl variants are not. The data strongly suggest that the LAC236 is implicated in the dimerization process of pro-LPH, most likely by nucleating the association of the ectodomains of the enzyme.
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PMID:Additional N-glycosylation and its impact on the folding of intestinal lactase-phlorizin hydrolase. 1074 59

The recent identification of two new thrombin receptors, PAR3 and PAR4, led us to re-examine the basis for endothelial cell responses to thrombin. Human umbilical vein endothelial cells (HUVEC) are known to express PAR1 and the trypsin/tryptase receptor, PAR2. Northern blots detected both of those receptors and, to a lesser extent, PAR3, but PAR4 message was undetectable and there was no response to PAR4 agonist peptides. To determine whether PAR3 or any other receptor contributes to thrombin signaling in HUVEC, PAR1 cleavage was blocked with two selective antibodies and PAR1 activation was inhibited with the antagonist, BMS200261. The antibodies completely inhibited HUVEC responses to thrombin, but BMS200261 was only partly effective, even though separate studies established that the antagonist completely inhibits PAR1 signaling at the concentrations used. Since peptides mimicking the PAR1 tethered ligand domain can also activate PAR2, we asked whether the remaining thrombin response in the presence of the antagonist could be due in part to the intermolecular transactivation of PAR2 by cleaved PAR1. Evidence that transactivation can occur was obtained in COS-7 cells co-expressing PAR2 and a variant of PAR1 that can be cleaved, but not signal. There was a substantial response to thrombin only in cells expressing both receptors. Conversely, in HUVEC, complete blockade of the thrombin response by the PAR1 antagonist occurred only when signaling through PAR2 was also blocked. From these observations we conclude that 1) PAR1 is the predominant thrombin receptor expressed in HUVEC and cleavage of PAR1 is required for endothelial cell responses to thrombin; 2) although PAR3 may be expressed, there is still no evidence that it mediates thrombin responses; 3) PAR4 is not expressed on HUVEC; and 4) transactivation of PAR2 by cleaved PAR1 can contribute to endothelial cell responses to thrombin, particularly when signaling through PAR1 is blocked. Such transactivation may limit the effectiveness of PAR1 antagonists, which compete with the tethered ligand domain rather than preventing PAR1 cleavage.
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PMID:Thrombin responses in human endothelial cells. Contributions from receptors other than PAR1 include the transactivation of PAR2 by thrombin-cleaved PAR1. 1078 64

In this study, we have investigated the distribution of the enzyme nucleoside triphosphate diphosphohydrolase-1 (NTPDase1; EC 3.6.1.5) in a subset of pig tissues by biochemical activity and Western blotting with antibodies against porcine NTPDase1. The highest expression of this enzyme was found in vascular endothelium, smooth muscle, spleen and lung. The complete cDNA of NTPDase1 from aorta endothelial cells was sequenced using primer walking. The protein consists of 510 amino acids, with a calculated molecular mass of 57 756 Da. The amino-acid sequence indicated seven putative N-glycosylation sites and one potential intracellular cGMP- and cAMP-dependent protein kinase phosphorylation site. As expected, the protein has a very high homology to other known mammalian ATPDases and CD39 molecules, and includes all five apyrase conserved regions. Expression of the complete cDNA in COS-7 cells confirmed that NTPDase1 codes for a transmembrane glycoprotein with ecto-ATPase and ecto-ADPase activities. Two proteolytic products of NTPDase1, with molecular mass of 54 and 27 kDa, respectively, were consistently present in proteins from transfected COS-7 cells and in particulate fractions from different tissues. A trypsin cleavage site, giving rise to these two cleavage products, was identified. In order to remain enzymatically active, the two cleavage products have to interact by non-covalent interactions.
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PMID:Distribution, cloning, and characterization of porcine nucleoside triphosphate diphosphohydrolase-1. 1086 13

The roles of N-linked glycosylation in the intracellular transport and fusion activity of the Sendai virus fusion (F) protein were studied. Each of three potential glycosylation motifs (designated g1, g2, and g3) in the F protein was mutated separately or in combination with the other sites. When the mutant F proteins were transiently expressed in COS cells, they showed significant changes in electrophoretic mobility, indicating that all three motifs in the F protein are glycosylated. Glycosylation-defective mutants which lacked the g2-oligosaccharide chain showed decreased immunoreactivity with a monoclonal antibody specific for the native conformation and were inefficiently transported to the cell surface. Such mutants, with the exception of a double mutant lacking g1 and g2-oligosaccharide chains, were also not able to induce syncytia formation when cells expressing them plus the hemagglutinin-neuraminidase protein were treated with trypsin. Mutations at the other glycosylation sites did not significantly affect the immunoreactivity with the monoclonal antibody or the efficiency of intracellular transport of the F protein. These results indicate that the N-linked oligosaccharide chain attached at g2 is important for efficient intracellular transport and for the fusion activity of the F protein.
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PMID:Functional analysis of the individual oligosaccharide chains of sendai virus fusion protein. 1087 59

Diacylglycerol kinases (DGKs) phosphorylate diacylglycerol produced during stimulus-induced phosphoinositide turnover and attenuate protein kinase C activation. Diacylglycerol kinase alpha is an 82-kDa DGK isoform that is activated in vitro by Ca(2+). The DGK alpha regulatory region includes tandem C1 protein kinase C homology domains and Ca(2+)-binding EF hand motifs. It also contains an N-terminal recoverin homology (RVH) domain that is related to the N termini of the recoverin family of neuronal calcium sensors. To probe the structural basis of Ca(2+) regulation, we expressed a series of DGK alpha deletions spanning its regulatory domain in COS-1 cells. Deletion of the RVH domain resulted in loss of Ca(2+)-dependent activation. Further deletion of the EF hands resulted in a constitutively active enzyme, suggesting that sequences in or near the EF hands are sufficient for autoinhibition. Binding of Ca(2+) to the EF hands protected sites within both the RVH domain and EF hands from trypsin cleavage and increased the phenyl-Sepharose binding of a recombinant DGK alpha fragment that included both the RVH domain and EF hands. These observations suggested that Ca(2+) elicits a concerted conformational change of these two domains. A cationic amphiphile, octadecyltrimethylammonium chloride, also activated DGK alpha. As with Ca(2+), this activation required the RVH domain. However, this agent did not protect the EF hands and RVH domain from trypsin cleavage. These findings indicate that the EF hands and RVH domain act as a functional unit during Ca(2+)-induced DGK alpha activation.
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PMID:A domain with homology to neuronal calcium sensors is required for calcium-dependent activation of diacylglycerol kinase alpha. 1091 59

An angiotensin II (AngII) peptidic analogue in which the third residue (valine) was substituted with the photoreactive p-benzoyl-L-phenylalanine (Bpa) was used to identify ligand-binding sites of the human AT(1) receptor. High-affinity binding of the analogue, (125)I-[Bpa(3)]AngII, to the AT(1) receptor heterologously expressed in COS-7 cells enabled us to efficiently photolabel the receptor. Chemical and enzymatic digestions of the (125)I-[Bpa(3)]AngII-AT(1) complex were performed, and receptor fragments were analyzed in order to define the region of the receptor with which the ligand interacts. Results show that CNBr hydrolysis of the photolabeled receptor gave a glycosylated fragment which, after PNGase-F digestion, migrated as a 11.4 kDa fragment, circumscribing the labeled domain between residues 143-243 of the AT(1) receptor. Digestion of the receptor-ligand complex with Endo Lys-C or trypsin followed by PNGase-F treatment yielded fragments of 7 and 4 kDa, defining the labeling site of (125)I-[Bpa(3)]AngII within residues 168-199 of the AT(1) receptor. Photolabeling of three mutant receptors in which selected residues adjacent to residue 168 were replaced by methionine within the 168-199 fragment (I172M, T175M, and I177M) followed by CNBr cleavage revealed that the bound photoligand (125)I-[Bpa(3)]AngII forms a covalent bond with the side chain of Met(172) of the second extracellular loop of the AT(1) receptor. These data coupled with previously obtained results enable us to propose a model whereby AngII adopts an extended beta-strand conformation when bound to the receptor and would orient itself within the binding domain by having its N-terminal portion interacting with the second extracellular loop and its C-terminus interacting with residues of the seventh transmembrane domain.
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PMID:Photolabeling identifies position 172 of the human AT(1) receptor as a ligand contact point: receptor-bound angiotensin II adopts an extended structure. 1093 82

The first step of papillomavirus infection is believed to be binding of major capsid protein L1 to the cell surface without involvement of minor capsid protein L2, but the viral infectivity can be neutralized either by anti-L1 or anti-L2 antibody. To understand the role of L2 in human papillomavirus (HPV) infection, we examined a segment of HPV type 16 (HPV16) L2, which contains a neutralization epitope common to HPV6, for its involvement in adsorption and penetration of the capsids. Preincubation of monkey COS-1 cells with a synthetic peptide having amino acids (aa) 108 to 120 of HPV16 L2 reduced the susceptibility of COS-1 cells to infection with HPV16 pseudovirions. Confocal microscopy showed that the green fluorescence protein (GFP) fused with the L2 peptide was found to bind to the surface of a HeLa cell, an HPV18-positive human cancer cell line, at 4 degrees C and to enter the cytoplasm after subsequent incubation at 37 degrees C. Flow cytometry showed that fused GFP did not bind to HeLa cells that had been treated with trypsin. Besides COS-1 and HeLa cells, some human and rodent cell lines were detected by flow cytometry to be susceptible to binding with fused GFP, showing a tendency of epithelial cells toward higher susceptibility. Substitutions at aa 108 to 111 inhibited fused GFP from binding to HeLa cells and reduced the infectivity in COS-1 cells of the in vitro-constructed pseudovirions. The results suggest that L2 plays an important role in enhancing HPV infection through interaction between the N-terminal region and a cellular surface protein, facilitating penetration of the virions and determining part of the tropism of HPVs.
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PMID:Human papillomavirus type 16 minor capsid protein l2 N-terminal region containing a common neutralization epitope binds to the cell surface and enters the cytoplasm. 1116 Jul 36


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