Cell surface anchorage and ligand-binding domains of the Saccharomyces cerevisiae cell adhesion protein alpha-agglutinin, a member of the immunoglobulin superfamily.

Abstract

a-Agglutinin is a cell adhesion glycoprotein expressed on the cell wall of Saccharomyces cerevisiae a cells. Binding of a-agglutinin to its ligand a-agglutinin, expressed by a cells, mediates cell-cell contact during mating. Analysis of truncations of the 650-amino-acid a-agglutinin structural gene AGed delineated functional domains of ea-agglutinin. Removal of the C-terminal hydrophobic sequence allowed efficient secretion of the protein and loss of cell surface attachment. This cell surface anchorage domain was necessary for linkage to a glycosyl phosphatidylinositol anchor. A construct expressing the N-terminal 350 amino acid residues retained full a-agglutinin-binding activity, localizing the binding domain to the N-terminal portion of e-agglutinin. A 278-residue N-terminal peptide was inactive; therefore, the binding domain includes residues between 278 and 350. The segment of a-agglutinin between amino acid residues 217 and 308 showed significant structural and sequence similarity to a consensus sequence for immunoglobulin superfamily variable-type domains. The similarity of the a-agglutinin-binding domain to mammalian cell adhesion proteins suggests that this structure is a highly conserved feature of adhesion proteins in diverse eukaryotes.Cell surface glycoproteins are involved in cell-cell adhesion in many eukaryotic systems. In the yeast Saccharomyces cerevisiae, haploid cells of a and a. mating types express adhesion glycoproteins called a-agglutinin and oa-agglutinin, respectively. Binding between these complementary cell wall proteins mediates aggregation of cells during mating (for reviews of agglutinin structure and function, see references 26 and 50). The agglutinins facilitate mating under conditions that do not promote cell-cell contact (28,35,38 (5,16,40,44).Both agglutinins are transported to the cell surface through the secretory pathway. Consistent with this finding, all three agglutinin structural genes, AGao1, AGA1, and AGA2, initiate with prototypical signal sequences for proteins that are transported through the secretory pathway (5, 16,28,35,42,43 (14). Therefore, we have speculated that GPI anchors are involved in cell surface localization of both agglutinins (26).To determine functional domains of oa-agglutinin, we have tested the effect of C-terminal truncations of AGot1. Our results delineate two independent domains. The C-terminal domain is required for cell surface anchorage, and the N-terminal half contains the binding domain of a-agglutinin. This N-terminal domain shows sequence and structural similarity to the immunoglobulin fold sequences found in many mammalian adhesion proteins, suggesting that the mechanism of adhesion mediated by glycoprotein-glycoprotein interactions is highly conserved. MATERLILS AND METHODSYeast strains. The agotl-J mutant (La2l), which is isogenic to wild-type strain W303-1B (MAToa ade2-1 his3-11,15 leu2-3,112 trpl-1 ura3-2 canl-100), was used to express the wildtype and truncated AGaoJ constructs, and the agcal::LEU2 strain used for immunoblots cont...

Identification of a ligand-binding site in an immunoglobulin fold domain of the Saccharomyces cerevisiae adhesion protein alpha-agglutinin.

“…Conserved residues are found within particular ,B strands, whereas the connecting loops are poorly conserved both with respect to sequence and length (Williams and Barclay, 1988). The similarity of a-agglutinin domain III to V-type Ig folds is comparable to the similarity between the Ig fold domains of CD4, CD8, Thyl, Po, and the CAM family (Wojciechowicz et al, 1993), and domain III is stabilized by a disulfide bond . Two additional a-agglutinin domains share structural features with Ig folds and significant sequence similarity to domain III Chen et al, 1995).…”

“…The C-terminal half consists of a highly glycosylated, serine/threonine-rich domain followed by a hydrophobic signal for attachment of a glycosyl phosphatidylinositol (GPI) anchor, which mediates anchorage to the cell wall (Wojciechowicz et al, 1993;Lu et al, 1994Lu et al, , 1995. The N-terminal half of a-agglutinin is active in binding to its ligand (Wojciechowicz et al, 1993); this adhesion region contains a domain (called domain III; Figure 1A) that shows similarity to immunoglobulin (Ig) fold domains (Williams and Barclay, 1988). Ig folds are characterized by a 13 barrel conformation containing either nine (Vtype) or seven (C-type) 13 strands assembled into two antiparallel 13 sheets.…”

Identification of SPT14/CWH6 as the yeast homologue of hPIG-A, a gene involved in the biosynthesis of GPI anchors

“…First, several wall proteins such as the sexual agglutinins and flocculin are known to contain a GPI anchor addition signal [2-41. Second, elongation of a -reporter protein with the C-terminal thirty amino acids of cY-agglutinin, which include the GPI anchor addition signal, is sufficient to target it to the cell wall [5]. Third, deletion of the GPI anchor addition signal from cu-agglutinin results in secretion of active a-agglutinin into the medium [6]. To further study the mechanism of cell wall protein incorporation, the Calcofluor white-hypersensitive cell wall mutant cwh6 [7] was chosen.…”

Identification and study of a Candida albicans protein homologous to Saccharomyces cerevisiae Ssr1p, an internal cell-wall protein

“…A Staab et al, 1999;Wojciechowicz et al, 1993). The presence of the GPI anchor seems to play an important role in the biology of C. albicans, as mutants are affected in morphogenesis, virulence and cell-wall composition (Richard et al, 2002).…”

“…Proteins of the Pir family can also be extracted from the cell wall by a mild NaOH treatment (Kapteyn et al, 1999;Mrsa et al, 1997). The presence of a GPI anchor has been Abbreviations: CR, Congo red; CW, calcofluor white; GPI, glycosylphosphatidylinositol; IPF, individual protein file.A Staab et al, 1999;Wojciechowicz et al, 1993). The presence of the GPI anchor seems to play an important role in the biology of C. albicans, as mutants are affected in morphogenesis, virulence and cell-wall composition (Richard et al, 2002).…”