Biochemical characterization of the binding region of Saccharomyces cerevisiae alpha-agglutinin: A member of the immunoglobulin superfamily.
Item
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Title
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Biochemical characterization of the binding region of Saccharomyces cerevisiae alpha-agglutinin: A member of the immunoglobulin superfamily.
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Identifier
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AAI9530859
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identifier
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9530859
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Creator
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Chen, Minhao Howard.
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Contributor
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Adviser: Peter N. Lipke
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Date
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1995
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Language
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English
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Publisher
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City University of New York.
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Subject
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Chemistry, Biochemistry | Biology, Microbiology | Health Sciences, Immunology | Biology, Molecular
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Abstract
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{dollar}\alpha{dollar}-agglutinin of S. cerevisiae is a glycosylated cell adhesion molecule that mediates cell adhesion during mating. The N-terminal 331 residues of {dollar}\alpha{dollar}-agglutinin ({dollar}\alpha{dollar}-agglutinin{dollar}\sb{lcub}20-351{rcub}{dollar}) is sufficient for binding to its ligand a-agglutinin. A region of {dollar}\alpha{dollar}-agglutinin{dollar}\sb{lcub}20-351{rcub}{dollar} with amino acid residues 220 to 300 showed significant homology to the consensus sequence of immunoglobulin variable-type (IgV) superfamily.;In this study, this previously described Ig-like region is expanded to residues 200 to 330 to accommodate all the potential {dollar}\beta{dollar} strands and the intradomain disulfide of an IgV domain. Two other regions, with amino acid residues 20-104 and 105-199, show significant sequence homology to each other and have sequence motifs common to IgV superfamily members. Therefore, {dollar}\alpha{dollar}-agglutinin{dollar}\sb{lcub}20-351{rcub}{dollar} consists of 3 IgV domains that together make up a binding environment for a-agglutinin. These regions are domain I: residues 20-104; domain II: residues 105-199; and domain III: residues 200-333.;{dollar}\alpha{dollar}-agglutinin{dollar}\sb{lcub}20-351{rcub}{dollar} was overexpressed and purified. The deN-glycosylated {dollar}\alpha{dollar}-agglutinin{dollar}\sb{lcub}20-351{rcub}{dollar} is a 45 kDa protein containing O-linked carbohydrates. Circular Dichroism spectroscopy showed a {dollar}\beta{dollar}-sheet content of 70% in {dollar}\alpha{dollar}-agglutinin{dollar}\sb{lcub}20-351{rcub}{dollar} and 55% in its proteolytic fragment {dollar}\alpha{dollar}-agglutinin{dollar}\sb{lcub}155-351{rcub}{dollar}. The high {dollar}\beta{dollar}-sheet content of {dollar}\alpha{dollar}-agglutinin{dollar}\sb{lcub}20-351{rcub}{dollar} confirms that the binding region of {dollar}\alpha{dollar}-agglutinin consists of anti-parallel {dollar}\beta{dollar}-sheet domains such as Ig-folds.;Proteolytic mixture containing fragments of amino acids 20-154 and 155-351 of {dollar}\alpha{dollar}-agglutinin{dollar}\sb{lcub}20-351{rcub}{dollar}, retaining the overall secondary structure of the binding region, shows no activity. Purified {dollar}\alpha{dollar}-agglutinin{dollar}\sb{lcub}155-351{rcub}{dollar} containing the entire domain III gives no activity, either. Therefore, the activity of {dollar}\alpha{dollar}-agglutinin is also dependent on structures of the first two domains.;The arrangement of the disulfide bonds in {dollar}\alpha{dollar}-agglutinin{dollar}\sb{lcub}20-351{rcub}{dollar} was established. Cys{dollar}\sp{97}{dollar} and Cys{dollar}\sp{lcub}114{rcub}{dollar} form a disulfide bond between domains I and II. Cys{dollar}\sp{lcub}202{rcub}{dollar} and Cys{dollar}\sp{lcub}300{rcub}{dollar} is an atypical intradomain disulfide bond between the A and F strands of domain III, whereas Cys{dollar}\sp{lcub}227{rcub}{dollar} and Cys{dollar}\sp{lcub}256{rcub}{dollar} have free sulfhydryls. Reduction of the disulfides is accompanied by a decrease in the {dollar}\beta{dollar}-sheet content and loss of the binding activity. Much of the primary structure of {dollar}\alpha{dollar}-agglutinin{dollar}\sb{lcub}20-351{rcub}{dollar} has been confirmed by peptide mapping, which revealed two N-glycosylation sites in domain III. In additional, a Ser and Thr rich region that cluster in the C-terminus of {dollar}\alpha{dollar}-agglutinin{dollar}\sb{lcub}20-351{rcub}{dollar} are highly O-glycosylated.
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Type
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dissertation
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Source
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PQT Legacy CUNY.xlsx
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degree
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Ph.D.
