Biomedical subjects

E P Chow

Publications and source records attributed to E P Chow.

18 recordsLinked to original sources

PubMed1992

Structure/function studies on vascular cell adhesion molecule-1.

Vascular cell adhesion molecule-1 (VCAM1) is a member of the immunoglobulin (Ig) superfamily which interacts with the integrin very late antigen-4 (VLA4). The VCAM1/VLA4 interaction mediates both adhesion and signal transduction and is thought to play an important role in inflammatory and immune responses in vivo. The major form of human VCAM1 contains seven extracellular Ig-like domains, with domain 1 designated as the most N-terminal. We have examined the relationship between human VCAM1 structure and function using a combination of domain truncation mutants and proteolytic fragmentation of recombinant soluble VCAM1. We have characterized two regions of VCAM1, localized to domains 4 and 5, which are highly sensitive to proteolytic cleavage, localized the epitope of the blocking monoclonal antibody 4B9 to domain 1, and found that domains 1-3 are sufficient for both its adhesive function and its ability to initiate T cell activation.

Amino Acid Sequence↗

PubMed1989

A dimeric form of lipocortin-1 in human placenta.

We have characterized a 68 kDa lipocortin from human placenta that was identified as a covalently linked homodimer of lipocortin-1 by peptide mapping and sequence analysis. The site of cross-linking was localized within the 3 kDa N-terminal tail region, an exposed domain that contains the phosphorylation sites for protein tyrosine kinase and protein kinase C and is sensitive to proteolysis. Sequence analysis of the corresponding peptide revealed that glutamine-18 was modified, suggesting that the cross-link may be generated by a transglutaminase. By incubating lipocortin-1 with placental membranes and with labelled glycine ethyl ester we observed a Ca2+-dependent labelling of lipocortin-1 within the tail region, supporting this notion. Like lipocortin-1, the dimer inhibits phospholipase Ad2 activity, is a substrate for the epidermal-growth-factor (EGF) receptor/kinase, and display Ca2+-dependent binding to phosphatidylserine-containing vesicles. In preparations from human placenta the dimer is particularly abundant, accounting for approx. 20% of the lipocortin-1.

Amino Acid Sequence↗

PubMed1989

Structure and properties of a human non-pancreatic phospholipase A2.

We have purified a human non-pancreatic phospholipase A2 that is present in platelets and is enriched in rheumatoid synovial fluid. The enzyme is calcium-dependent, has a pH optimum of 8-10, and shows a striking preference for substrate presented in the form of Escherichia coli membranes. In the E. coli phospholipase A2 assay the phospholipase exhibits an apparent specific activity of 300 mumol/mg/min. Using oligonucleotide probes based on amino-terminal sequence data, we cloned the corresponding human gene from a genomic DNA library and expressed the gene in animal cells. The protein was secreted from the cells in an active form. The deduced amino acid sequence of the human protein consists of 124 amino acids, contains structural features common to all known phospholipase A2s, and has a half-cystine pattern that is characteristic of the snake venom group II enzymes.

Amino Acid Sequence↗

PubMed1989

Agkistrodon piscivorus piscivorus platelet aggregation inhibitor: a potent inhibitor of platelet activation.

Applaggin (Agkistrodon piscivorus piscivorus platelet aggregation inhibitor) is a potent inhibitor of platelet activation. The protein is isolated from the venom of the North American water moccasin snake in three steps, including gel filtration, cation exchange, and reverse-phase HPLC procedures. The purified protein migrates as a 17,700-Da polypeptide by SDS/PAGE under nonreducing conditions and as a 9800-Da peptide in the presence of thiol. The behavior of applaggin on SDS/PAGE would indicate that the protein is a disulfide-linked dimer. Applaggin has been completely sequenced by Edman degradation and consists of 71 amino acids. The sequence is rich in cysteine and contains Arg-Gly-Asp at residues 50-52. Applaggin blocks platelet aggregation induced by ADP, collagen, thrombin, or arachidonic acid with IC50 values ranging from 12 to 128 nM (0.2-2.3 micrograms/ml) depending on the agonist and its concentration. This inhibition is found to correlate with inhibition of thromboxane A2 generation and of dense granule release of serotonin. Inhibition by applaggin of serotonin release induced by ADP, gamma-thrombin, and collagen was monitored in plasma under stirred conditions with [3H]serotonin-loaded platelets, and IC50 values for inhibition are found to range from less than 10 to 145 nM. At saturating concentrations, 125I-labeled applaggin (125I-applaggin) binds to 28,500 sites per unstimulated, washed platelet with a Kd of 1.22 x 10(-7) M. Binding of 125I-applaggin to platelets is inhibited by the synthetic undecapeptide Arg8-Gly-Asp-Val at 200 microM.

Adenosine Diphosphate↗

PubMed1989

Structural and functional division into two domains of the large (100- to 115-kDa) chains of the clathrin-associated protein complex AP-2.

The clathrin-associated protein complex 2 (AP-2 complex) is a group of proteins associated with clathrin-coated vesicles and believed to interact with cytoplasmic domains of receptors found in the plasma membrane. AP-2 was purified as an assembly of several polypeptide chains (alpha, beta, AP50, and AP17), of which only the alpha and beta chains (100-115 kDa) show significant heterogeneity. We have obtained cDNA clones for two distinct rat brain beta chains. We have also studied the domain organization of bovine brain AP-2 complexes by selective proteolysis. Results of these studies show that the alpha and beta chains have a similar two-domain organization. Their amino-terminal domains are relatively invariant whereas their carboxyl-terminal domains are variable in both sequence and length. We propose that the variable domains select receptors for inclusion in coated vesicles.

Adaptor Protein Complex 2↗

PubMed1988

Proteolytic processing of mullerian inhibiting substance produces a transforming growth factor-beta-like fragment.

Mullerian inhibiting substance (MIS) is a differentiation factor that causes the Mullerian duct to regress during the development of the male reproductive tract. The active form is a disulfide-linked dimer consisting of two identical 70-kDa subunits. Recently, the amino acid sequence for MIS was deduced from its gene sequence and revealed that the carboxyl-terminal region shares homology with transforming growth factor (TGF)-beta. Since TGF-beta is produced as a large latent precursor that requires proteolytic activation for activity, we sought to determine if MIS might undergo a similar processing event. Here we demonstrate that typically 5 to 20% of the protein in MIS preparations is cleaved at a site 109 amino acids from the carboxyl terminus. Concurrent cleavages from both chains of the MIS dimer produces a 25-kDa TGF-beta-like fragment and a high molecular mass complex derived from the amino terminus of the protein. Although the two fragments are noncovalently linked, they remain tightly associated after cleavage, and thus are structurally organized like TGF-beta within its precursor. The same cleavage products also can be generated by limited proteolysis with plasmin, which provides a simple method for converting the entire preparation into the cleaved form. The plasmin-digested MIS is fully active in the organ culture assay.

Amino Acids↗

PubMed1988

Five distinct calcium and phospholipid binding proteins share homology with lipocortin I.

We have purified two 35-kDa proteins from rat peritoneal lavages that inhibit phospholipase A2 activity. Both are calcium/phospholipid-dependent membrane binding proteins and share similar structural and biochemical properties with lipocortins I and II. By sequence analysis we confirmed that they are lipocortin-related, and we refer to the two inhibitors as lipocortins III and V. Using partial sequence information obtained from the purified rat proteins, full length cDNA clones for both proteins and for their human counterparts were isolated. As with lipocortins I and II, the amino acid sequences of lipocortins III and V which were deduced from the cDNA clones are highly conserved, sharing 50% identity with other family members. Related proteins were also purified from bovine intestinal mucosa and characterized by peptide mapping, sequence, and immunological analyses. In addition to lipocortins III and V the bovine preparation contained a third 35-kDa inhibitor and a 68-kDa inhibitor, extending the number of known lipocortins to six distinct proteins. While the various lipocortins are structurally similar, distinct differences in their cellular distribution indicate specialized roles for the individual proteins.

Amino Acid Sequence↗

PubMed1988

Location of sites in human lipocortin I that are phosphorylated by protein tyrosine kinases and protein kinases A and C.

Lipocortin I is a 39-kilodalton membrane-associated protein that in A431 cells is phosphorylated on tyrosine in response to epidermal growth factor (EGF). We have used recombinant human lipocortin I as a substrate for several protein kinases and identified phosphorylated residues by a combination of peptide mapping and sequence analysis. Lipocortin I was phosphorylated near the amino terminus at Tyr-21 by recombinant pp60c-src. The same tyrosine residue was phosphorylated by polyoma middle T/pp60c-src complex, by recombinant pp50v-abl, and with A431 cell membranes by the EGF receptor/kinase. The primary site of phosphorylation by protein kinase C was also near the amino terminus at Ser-27. The major site of phosphorylation by adenosine cyclic 3',5'-phosphate dependent protein kinase was on the carboxy-terminal half of the molecule at Thr-216. These sites are compared to the phosphorylation sites previously located in the structurally related protein lipocortin II.

Amino Acid Sequence↗

PubMed1988

Molecular cloning and complete amino acid sequence of AP50, an assembly protein associated with clathrin-coated vesicles.

AP50 is the 50,000-dalton protein component found in clathrin-coated vesicles as part of the coat assembly protein (AP) complex, AP-2. AP50 cDNA clones were isolated from rat brain cDNA libraries, and their nucleotide sequence was determined. The isolated cDNA clones represent the entire coding sequence for the rat brain AP50. They encode a polypeptide containing 435 amino acids with a molecular weight of 49,612 daltons. Comparison with the partially sequenced bovine brain AP50 shows a primary structure that is highly conserved. AP50 does not have detectable sequence similarity with other known kinases or with other proteins of known sequence.

Adaptor Protein Complex 2↗

PubMed1987

Purification and characterization of proteolytic fragments of lipocortin I that inhibit phospholipase A2.

Human lipocortin I is a 38.5-kDa phospholipase A2 inhibitor that has been produced in Escherichia coli in large quantities by recombinant DNA technology (Wallner, B.P., Mattaliano, R.J., Hession, C., Cate, R. L., Tizard, R., Sinclair, L.K., Foeller, C., Chow, E.P., Browning, J.L., Ramachandran, K.L., and Pepinsky, R.B. (1986) Nature 320, 77-80). To localize the region within the protein responsible for its inhibitory activity, we generated a series of fragments of the recombinant product by limited proteolysis with elastase and characterized their structure by sequencing and peptide mapping. Five active fragments have been analyzed in detail. The smallest is an 18-kDa fragment derived from the amino-terminal half of lipocortin. Three of the larger fragments contain this region. The fifth fragment is missing 83 amino acids from the amino terminus. A region common to all the active fragments (amino acid residues 97-178) is 70% homologous with the corresponding region from a second member of the lipocortin family which recently was cloned (Huang, K-S., Wallner, B.P., Mattaliano, R.J., Tizard, R., Burne, C., Frey, A., Hession, C., McGray, P., Sinclair, L.K., Chow, E.P., Browning, J.L., Ramachandran, K.L., Tang, J., Smart, J.E., and Pepinsky, R.B. (1986) Cell 46, 191-199) and thus presumably is important for activity. In addition to inhibitory fragments, we have isolated a 3-kDa proteolytic fragment from the amino terminus of lipocortin I that contains the known phosphorylation site for protein-tyrosine kinases. Because of sequence homology of the 3-kDa fragment with biologically active synthetic peptides from pp60v-src and middle T antigen, its release by proteases may represent an important part of the activity of lipocortin.

Amino Acid Sequence↗

PubMed1987

Clathrin light chains LCA and LCB are similar, polymorphic, and share repeated heptad motifs.

The clathrin light chains fall into two major classes, LCA and LCB. In an intact clathrin triskelion, one light chain, of either class, is bound to the proximal segment of a heavy chain leg. Analysis of rat brain and liver complementary DNA clones for LCA and LCB shows that the two light chain classes are closely related. There appear to be several members of each class having deletions of varying length aligned at the same position. A set of ten heptad elements, characteristic of alpha-helical coiled coils, is a striking feature of the central part of each derived amino acid sequence. These observations suggest a model in which the alpha-helical segment mediates binding to clathrin heavy chains and the amino- and carboxyl-terminal segments mediate interactions with other proteins. They also suggest an explanation for the observed tissue-dependent size variation for members of each class.

Amino Acid Sequence↗

PubMed1987

Clathrin heavy chain: molecular cloning and complete primary structure.

We have deduced the 1675-amino acid sequence of rat clathrin heavy chain from cDNA clones and predict a protein of Mr 191,569. We have established the polarity of the heavy chain and assigned sequence positions to several structural landmarks of the clathrin leg. The terminal domain at the distal end of the clathrin leg is at the amino terminus of the heavy chain. It is connected to the distal segment by a flexible "link" from Tyr-479 to Arg-523. There is an unusual sequence at the carboxyl terminus that may form the globular projection at the vertex of the clathrin trimer. We suggest that a possible site of heavy-chain-light-chain interaction is located in the proximal segment. Comparison with other partially sequenced mammalian clathrin heavy chains shows that the primary structure is highly conserved. The heavy chain is unrelated to other classes of structural proteins.

Amino Acid Sequence↗

PubMed1986

Structure of turnip crinkle virus. III. Identification of a unique coat protein dimer.

The minor structural protein (p80), found in about one copy per virion in turnip crinkle virus (TCV), is shown by amino acid analysis and peptide mapping to be a covalent dimer of the major coat protein (p40). The covalent linkage occurs near the N termini of the crosslinked chains. These data suggest that TGV and related viruses contain 178 copies of p40 (89 non-covalent dimers) and one copy of p80 (covalent dimer of two additional p40 chains). The presence of p80 in the salt-stable RNA-protein complex formed when TCV dissociates, as described in an accompanying paper, indicates that the covalent modification affects binding to RNA. We suggest that p80 might be the final dimer to be incorporated into the shell and that it might also be the site for initiation of uncoating.

Amino Acids↗

PubMed1986

Two human 35 kd inhibitors of phospholipase A2 are related to substrates of pp60v-src and of the epidermal growth factor receptor/kinase.

We have purified two 35 kd phospholipase A2 inhibitors from human placenta, which we refer to as lipocortin I and II. Both proteins exhibit similar biochemical properties and occur in placenta at about 0.2% of the total protein. By peptide mapping, sequence, and immunological analyses, we show that lipocortin I and the 35 kd substrate for the EGF-receptor/kinase from A431 cells are the same protein. By similar criteria, we determine that lipocortin II is the human analogue of pp36, a major substrate for pp60src, which has been characterized in chicken embryo fibroblasts and in bovine brush border preparations. The amino acid sequences of lipocortin I and II that we deduced from cDNA clones share 50% homology, indicating that they probably evolved from a common gene.

Amino Acid Sequence↗

PubMed1986

Isolation of the bovine and human genes for Müllerian inhibiting substance and expression of the human gene in animal cells.

We have isolated the bovine and human genes for Müllerian inhibiting substance (MIS), a testicular glycoprotein that causes regression of the Müllerian duct during development of the male embryo. The mRNA sequence of bovine MIS, determined from an analysis of cDNA and genomic clones, codes for a protein of 575 amino acids containing a 24 amino acid leader peptide. The human gene has five exons that code for a protein of 560 amino acids. A comparison of the bovine and human MIS proteins reveals a highly conserved C-terminal domain that shows marked homology with human transforming growth factor-beta and the beta chain of porcine inhibin. Animal cells transfected with the human gene secrete biologically active MIS, which causes regression of the rat Müllerian duct in vitro.

Amino Acid Sequence↗

PubMed1986

Disulfide scrambling of interleukin-2: HPLC resolution of the three possible isomers.

Native interleukin-2 (IL-2) contains three cysteines; two exist in a disulfide bridge (Cys-58 and Cys-105) and the third Cys-125 is a free sulfhydryl. In the presence of 6 M guanidine hydrochloride at alkaline pH, IL-2 is converted into three isomers. Each isomer represents one of the three possible disulfide-linked forms that can be generated from three cysteines. These three isomers were resolved on a C4 reverse-phase HPLC system. The identity of each of the three forms was determined by carboxymethylation of the free cysteines in each isomer with [3H]iodoacetic acid followed by determination of the labelled cysteines by tryptic peptide mapping. Tryptic peptide mapping of the more predominant of the two scrambled peaks showed it to be the Cys-105-S-S-Cys-125 linked form of IL-2. A Ser-125 construction of IL-2, which lacks a free cysteine, did not scramble under these conditions. These experiments demonstrate the utility of reverse-phase HPLC in studies of protein folding and disulfide bond structure.

Amino Acid Sequence↗

PubMed1986

Purification and partial sequence analysis of a 37-kDa protein that inhibits phospholipase A2 activity from rat peritoneal exudates.

We have purified from rat peritoneal exudates a 37-kDa protein that inhibits phospholipase A2 activity. It is the predominant phospholipase inhibitor protein in these preparations and also is detected in a wide variety of cell lines. Levels of expression range from 0 to 0.5% of total protein. In the peritoneal preparations, the inhibitor is partially proteolyzed into a series of lower mass forms, including species at 30, 24, and 15 kDa. These fragments all are immunoreactive with an antibody raised against the 37-kDa protein. The rat protein also is immunoreactive with an antibody developed against a 6-kDa phospholipase inhibitor protein from snake venom. The primary structure of more than half of the rat inhibitor has been deduced by protein microsequence analysis. These sequences are closely related to sequences from its human analogue, which we recently cloned and expressed (Wallner, B. P., Mattaliano, R. J., Hession, C., Cate, R. L., Tizard, R., Sinclair, L. K., Foeller, C., Chow, E. P., Browning, J. L., Ramachandran, K. L., and Pepinsky, R. B. (1986) Nature, in press), and thus we infer that the inhibitor is highly conserved evolutionarily. Properties of the molecule suggest that it is a member of a family of steroid-induced anti-inflammatory proteins collectively referred to as lipocortin.

Amino Acid Sequence↗

PubMedDate not supplied

Cloning and expression of human lipocortin, a phospholipase A2 inhibitor with potential anti-inflammatory activity.

The anti-inflammatory action of glucocorticoids has been attributed to the induction of a group of phospholipase A2 inhibitory proteins, collectively called lipocortin. These proteins are thought to control the biosynthesis of the potent mediators of inflammation, prostaglandins and leukotrienes, by inhibiting the release of their common precursor, arachidonic acid, a process that requires phospholipase A2 hydrolysis of phospholipids. Lipocortin-like proteins have been isolated from various cell types, including monocytes, neutrophils and renal medullary cell preparations. The predominant active form is a protein with an apparent relative molecular mass (Mr) of 40,000 (40K). These partially purified preparations of lipocortin mimic the effect of steroids, and mediate anti-inflammatory activity in various in vivo model systems. Using amino-acid sequence information obtained from purified rat lipocortin, we have now cloned human lipocortin complementary DNA and expressed the gene in Escherichia coli. Our studies confirm that lipocortin is a potent inhibitor of phospholipase A2 activity.

Amino Acid Sequence↗