Sexual agglutinins are expressed on the surface of haploid budding yeasts, including Saccharomyces cerevisiae (Lipke and Kurjan, 1992; Pierce and Ballou, 1983; Hagiya et al., 1977; Crandall et al., 1974; Crandall and Brock, 1968). During mating, the interaction of complementary agglutinins of each species mediates direct cell-cell contact to promote fusion of pairs of mating partners to form diploid zygotes. Mutants defective in these sexual agglutinins are mating-deficient in liquid medium (Lipke et al., 1989).

S. cerevisiae -agglutinin is a highly glycosylated cell wall-anchored protein that is constitutively expressed on cells of the mating type and is induced to greater expression levels in response to the mating pheromone, a-factor (Terrance et al., 1987; Hauser and Tanner, 1989; Lipke et al., 1989). The open reading frame of the -agglutinin gene, AG1, encodes a single polypeptide of 650 amino acids, including an NH-terminal secretion signal (residues 1-19) and a COOH-terminal glycosylphosphatidylinositol (GPI) ()addition signal that is involved in cell wall anchorage (residues 628-650) (Kodukula et al., 1993; Wojciechowicz et al., 1993; Kapteyn et al., 1994; Lu et al., 1994, 1995; Van Berkel et al., 1994). The NH-terminal part of the mature protein (residues 20-350) contains the binding region, which has been proposed to consist of three domains (Wojciechowicz et al., 1993). These features are summarized in Fig. 1.

Figure 1: Features of the -agglutinin sequence. The open reading frame of the Ag1 gene is shown. The NH-terminal secretion signal and the COOH-terminal GPI addition signal are colored solid black. Proposed IgV domains and the Ser/Thr-rich sequence are marked.

Within the NH-terminal half, a segment (amino acid residues 200-326, designated domain III) shows significant similarity to variable domains of the immunoglobulin superfamily (IgV domains) based on the amino acid sequence and predicted -sheet profile analysis (Wojciechowicz et al., 1993). A His residue essential for binding has been identified within this putative domain (Cappellaro et al. 1991), and other essential residues have been identified by site-specific mutagenesis. ()We have proposed that domains I and II are also Ig-like, but evidence to support this contention has been lacking.

In Ig domains, post-translational modifications help determine tertiary structure (Dwek et al., 1993; Williams and Barclay, 1988). We have investigated the disulfide bonding pattern of the 6 Cys residues and the positions of the N- and O-glycosylations in the Ig-like region (Terrance et al., 1987; Hauser and Tanner, 1989). N-Linked glycans are not important for cell adhesion, because endo H treatment or synthesis in the presence of tunicamycin does not affect binding activity (Terrance et al., 1987). O-Linked glycans are also present and appear to account for a significant portion of the apparent size of -agglutinin (Wojciechowicz et al., 1993; Lu et al., 1994).

We have now produced a 332-residue active fragment, -agglutinin, in quantities sufficient to allow investigation of the secondary structure and determine the positions of post-translational modifications. The results, along with those of a modified sequence alignment procedure, result in a model for -agglutinin.

EXPERIMENTAL PROCEDURES

Chemicals and Reagents

Yeast Strains and Expression Vector

Construction of pPGK-AG1

Overexpression and Purification of -Agglutininfrom Culture Supernatant

The active material was dialyzed and lyophilized. The dry powder was resuspended in 10 mM potassium chloride, 10 mM sodium acetate, pH 5.5, 0.01% SDS, and 1 mM EDTA and incubated with 1:200 to 1:500 molar ratio of endo H for 4-6 h at 25 °C or overnight at 4 °C. Under these conditions, there was no detectable proteolysis of the -agglutinin. The de-N-glycosylated -agglutinin was chromatographed on a Bio-Gel P-60 size exclusion column (60-ml bed volume) which had been previously equilibrated with 30 mM sodium acetate buffer, pH 5.5.

Immunoblots

CD and Structure Analysis

Endoprotease Digestions

HPLC

Cysteine-specific Labeling of Tryptic Peptides of -Agglutinin

NH-terminal Sequencing of Peptides

Figure 8: Summary of sequenced -agglutinin peptides. Regions sequenced from with tryptic and S. aureus V8 peptides are underlined with solid or wavy lines, respectively. Sulfhydryl groups are labeled (SH) and disulfide bonds are marked. Identified O-linked glycosylation sites are marked (solid diamonds). Potential N-glycosylation sites are italicized and stricken out; the two identified N-glycosylation sites are marked (stacked solid diamonds).

Dot Blot Analysis of O-Linked Proteolytic -AgglutininPeptides

Other Methods

RESULTS

Expression and Secretion of -Agglutinin

Figure 2: Immunoblot of -agglutinin from culture supernatant. Supernatant from a culture of L21 [pPGK-AG1] (17 ml) was lyophilized to dryness, resuspended in 200 µl of distilled water, and passed through a Bio-Gel P-10 column preequilibrated with 0.01 M sodium acetate, pH 5.5. Desalted material (20 µl) was treated without or with endo H (0.5 µl of 1 unit/ml) at room temperature for 2 h. Samples without and with Endo H treatment were analyzed by electrophoresis in the absence or presence of the reducing reagent DTT as indicated.

Figure 3: Bio-Gel P-60 chromatography of endo H-treated -agglutinin. The active material from DEAE-Sephadex A-25 was lyophilized to dryness. The material was resuspended and dialyzed against 0.03 M sodium acetate, pH 5.5, treated with endo H (15 µl of 1 unit/ml endo H to 2000 units of -agglutinin activity) and loaded onto a Bio-Gel P-60 column preequilibrated with the same buffer. Fractions (3 ml) were collected and monitored at 280 nm (A). Aliquots of fractions were electrophoresed on a 12% SDS-PAGE gel and visualized by staining with Coomassie Blue (B). Molecular size markers are shown on the left.

Elution of endo H-treated -agglutinin from a Bio-Gel P-60 column gave purified -agglutinin with an apparent molecular size of 45 kDa for the smaller species on SDS gels (Fig. 3). The deduced M of -agglutinin from the predicted amino acid sequence is 37,108.

Therefore, N-linked carbohydrate accounts for two-thirds of the apparent 110-kDa molecular mass of -agglutinin, and the O-linked carbohydrate remaining after endo H digestion could account for an additional 8 kDa of apparent mass.

Endoprotease Arg-C Digestion of -Agglutinin

Figure 4: SDS-PAGE analysis of endoprotease Arg-C-digested -agglutinin. Samples of endoprotease Arg-C-digested -agglutinin (left lanes) and endoprotease alone (center lanes, labeled ``enzyme'') were treated with or without DTT as marked, electrophoresed on a 15% SDS-polyacrylamide gel, and the gel was stained with Coomassie Blue. Molecular size standards on the right were from 97,400 to 4000 Da.

The NH-terminal sequence of each fragment was determined by microsequence analysis after electroblotting onto polyvinylidene difluoride membranes. Both the 16- and 21-kDa fragments had the same NH-terminal sequence as mature -agglutinin, beginning at Ile, immediately following the secretion signal sequence (Table 1). The 21-kDa form represented a species with some N-linked carbohydrate remaining and generated a 16-kDa fragment after additional treatment with endo H (data not shown). The NH-terminal -agglutinin polypeptide from Ile to Lys would have a molecular mass of 15,119 daltons, close to the value for the 16-kDa peptide. The 31-kDa fragment, called -agglutinin, started with Ser-Gly-Pro-Met-Leu-Val (Table 1). The predicted molecular mass of this peptide is 21,989 Da. The extra 7 kDa of apparent molecular mass in agglutinin may be attributed to the presence of multiple O-glycosylations (see below). No additional fragments were seen, including any of the predicted peptides following Arg residues (Fig. 4). Therefore, endoprotease Arg-C cleaved only at Lys, instead of any of the six Arg residues in -agglutinin.

Endoprotease Arg-C from Clostridium histolyticum also cleaved at Lys only (data not shown). Peptide sequencing confirmed that the cleaved residue was Lys. No fragments were generated in -agglutinin incubated without protease. Therefore, hydrolysis of -agglutinin at Lys was endoprotease Arg-C specific and not due to proteolytic activity in the -agglutinin preparations or in other reagents used for the digestion. Tosyl-lysyl chloroketone inhibits Arg-C (Mazzoni et al., 1991); therefore, Arg-C must have proteolytic activity toward Lys.

Agglutination Activity of Proteolytic Fragments of -Agglutinin

CD of Native -Agglutinin

Figure 5: Far-UV CD spectra of -agglutinin. Each spectrum represents the average of five individual spectra taken at 1.0-nm intervals as specified under ``Experimental Procedures.'' Equivalent molar concentration of each sample were examined. Spectra of native (solid line) -agglutinin and endoprotease Arg-C-digested -agglutinin (dashed line).

CD of -AgglutininDigested with Endoprotease Arg-C

Disulfides in Endoprotease Arg-C-digested -Agglutinin

Identification of Disulfide Bonds

-Agglutinin was digested with trypsin in the presence or absence of DTT, and the products were separated by reversed phase chromatography on a C18 column. Three tryptic peptides (T1, T2, T2`) were unique to the nonreduced chromatogram (Fig. 6A), and three peptides (DT1, DT2, and DT3) were unique to the reduced chromatogram (Fig. 6B). These peptides were sequenced and compared with the sequences of the Cys-containing tryptic fragments predicted from the gene sequence (Table 2Table 3Table 4). Peaks T1 and DT1 had the sequence of the predicted peptide containing both Cys and Cys. As with the change in gel mobility, the change in retention time in the presence of DTT implied that these two Cys residues formed an internal disulfide. Similar chromatography and sequencing analyses of peptides from S. aureus V8 digests confirmed this assignment ( Table 3and Table 4): peptide DS2 was seen only after reduction and contained Cys. As expected, tryptic peptide T1 containing Cys and Cys was labeled with P-2007 after reduction, but was not labeled in nonreduced samples (Fig. 7, A and B).

Figure 6: Chromatogram of reduced and nonreduced trypsin-digested -agglutinin. Mixtures of trypsin digested peptides treated without (A) or with DTT (B) were chromatographed. Peaks unique to the nonreduced (T1, T2, T2`), and reduced (DT1-DT3) profiles are labeled. The peptide containing Cys and Cys is peak T4 in nonreduced and peak DT4 in the reduced profile. The amino acid sequences of these peptides are listed in Table 1, Table 2, Table 3, and Table 4. Both chromatograms were obtained under standard conditions, and the retention times shown in B apply to both chromatograms. Fraction numbers shown in A correspond to those mentioned in the text for concanavalin A blotting.

Figure 7: HPLC chromatograms of P-2007-labeled tryptic -agglutinin peptides. Tryptic peptides labeled with P-2007 in the absence (A) or presence (B) of the reducing reagent TCP were fractionated by reversed phase HPLC using the standard program, and the eluant was monitored at 340 nm. Peptide T4, containing Cys and Cys, was labeled under nonreduced conditions, isolated (C), digested with endoprotease Asn-N, and rechromatographed (Panel D).

Tryptic peaks T2 and T2` each yielded two sequences in approximately equimolar amounts ( Table 2and Table 4). These sequences were those expected for disulfide-linked peptides containing Cys and Cys. Note that the peptides containing Cys do not contain Cys, because Lys is efficiently cleaved (Fig. 6; Table 4). The difference in retention times of T2 and T2` must be due to differential modification of the fragments; differences in the extent of glycosylation of the peptide fragment containing Cys would yield this result. In the chromatogram of tryptic peptides from reduced -agglutinin, peaks T2 and T2` were absent, and new peaks appeared with retention times of 117 and 154 min (labeled DT2 and DT3 in Fig. 6B). Sequencing showed that these peaks were peptides predicted to include Cys and Cys, respectively. These results show that Cys and Cys are disulfide bonded. Sequencing of S. aureus V8-digested peptides ( Table 3and Table 4) and P-2007 labeling (Fig. 7) also confirmed this result.

Cysand CysHave Free Sulfhydryls

To verify that peptide peak T4 in the nonreduced profile contained Cys and Cys as free sulfhydryls, this peptide was labeled with P-2007. This peptide alone was labeled in reactions of tryptic digests with P-2007 under nonreducing conditions (Fig. 7, A versus B). To determine if the peptide contained two labeled cysteines, the isolated labeled peptide (Fig. 7C) was further digested with endoprotease Asp-N and rechromatographed (Fig. 7D). Two additional labeled peptides were detected at 35 and 45 min, as a result of the digestion. These peptides had the retention times expected for the labeled peptides containing Cys and Cys, respectively. The original labeled peptide with a retention time of 53 min, however, was still present, probably due to incomplete digestion. Therefore, both Cys and Cys are free cysteines.

Identification of O-Linked Glycosylation Sites by Peptide Sequencing

Eight Ser residues (positions 282, 316, 331, 334, 335, 338, 346, and 350) and 15 Thr residues (positions 289, 299, 303, 307, 308, 311, 314, 315, 329, 339, 340, 341, 342, 345, and 349) were found to be modified in tryptic peptides and/or S. aureus V8 peptides (Table 6). Therefore, all of the eight Ser and 15 Thr residues from Ser to the COOH terminus of -agglutinin were modified. All other sequenced Ser and Thr residues were observed as expected (Fig. 8).

Confirmation of O-Glycans with ConA

Identification of N-Linked Glycosylation Sites in -Agglutinin

DISCUSSION

-Agglutinin is fully active and must therefore form a correctly folded structure. A high proportion of -sheet structure is present throughout the protein. Thus, physical evidence bolsters sequence similarity arguments that there are three IgV-like domains in -agglutinin.

Domains of -Agglutinin

Figure 9: Alignment of three domains of -agglutinin with each other and with a consensus sequence for IgV domains (Williams and Barclay, 1988). The positions of the -strands in the consensus sequence are shown. The alignment is based on secondary structure prediction and alignment within prospective -strands, with gaps allowed only between strands (Chou and Fasman; Lipke et al., 1995). The sequence between residues 101 and 110 is repeated as the G strand of domain I and the A strand of domain II, as discussed in the text. Identities are boxed and shaded, similarities are boxed without shading. Similarity sets are: A, F, I, L, M, V, Y; A, G; C, S, P; D, E; D, N; E, Q; H, K, R; H, W, Y; N, Q; S, T;. represents a hydrophobic residue in the consensus and includes A, F, I, L, M, P, V, Y, and W.

Assignment of domain III as an IgV-like domain suggests that there may be additional Ig-like domains in the NH-terminal region, because multiple sequential Ig domains are often present in members of the Ig superfamily. In members of the superfamily that are cell adhesion proteins, 2 to 5 sequential domains are common. These tandem domains are at the NH termini of the mature proteins in the vast majority of cases (Williams and Barclay, 1988). Furthermore, the Ig fold appears to be more widespread than the Ig superfamily itself and proteins with little or no sequence similarity to Ig domains form Ig-like folds. Most of these proteins are involved in cell adhesion or protein-protein interaction (Holmgren et al., 1992; Overduin et al., 1995; Shapiro et al., 1995).

The 180 NH-terminal residues of -agglutinin are enough to form two more IgV domains, with the G strand of domain I being the A strand of domain II, as in CD4 (Fig. 9) (Williams and Barclay, 1988; Williams et al., 1989; Ryu et al., 1990; Wang et al., 1990; Barclay et al., 1993). A revised alignment procedure for -agglutinin strongly supports a three-domain assignment (Fig. 9) (Lipke et al., 1995). When the sequences of the three proposed domains were aligned with each other and with an IgV consensus based on predicted strand profile (Fig. 9) and hydrophobic moment (Eisenberg et al., 1984) (data not shown), there was high conformity to the consensus in all three domains (Table 7). Although there is a low degree of identity in the alignment, the conserved residues include many of the IgV consensus residues. The alignments shown scored significantly better (Z > 3) than did random sequences of the same composition. Residues in -agglutininin domains I and II corresponding to the consensus positions for the IgV domains include a Cys residue in each domain (the F strand Cys in domain I and the B strand Cys in domain II) and Trp corresponding to strand C of domains I. There are Met residues in all three proposed -agglutinin domains in positions analogous to the conserved D-strand Arg in other IgV domains (residues 69, 158, and 274, Fig. 9). In IgV domains, an Asp residue at the beginning of the F strand forms a salt bridge with this Arg, which it could not do with the Met residue in the -agglutinin. In the three proposed -agglutinin domains, this Asp is also absent (residues 89, 176, and 293). Although the number of residues conserved among the three domain is low, the three sequences show about 40% similarity (Table 7). The conserved and identical residues are especially frequent at positions conserved in mammalian IgV domains ( Fig. 9and Table 7).

The similarity of domains I and II is also consistent with apparent sequence homology by a standard method. Residues 30-94 and 107-180 can be aligned with a Z score of 4.7 (GCG BESFIT, gap weight 3.0, length weight 0.0; Gribskov and Devereux, 1991). Such a score implies a common ancestral sequence and common structure for these regions, which correspond to strands B to F of domains I and II.

CD Spectra Are Consistent with Inclusion of -Agglutinin in the Ig Superfamily

Domain III (residues 200-326) was previously proposed to contribute to the binding site (Cappellaro et al., 1991; Lipke and Kurjan, 1992). Neither the purified -agglutinin fragment nor the unpurified Arg-C digest of -agglutinin retained activity, despite the retention of most of the secondary structure in the cleaved product. The inactivity of the cleaved product implies that regions of domains I and/or II are also essential for binding. Such contributions of multiple domains to the binding site is the rule in the Ig superfamily, with few exceptions (Williams and Barclay, 1988).

Disulfide Bonds and Free Sulfhydryls in -Agglutinin

Figure 10: Structure of -agglutinin. The standard ``C''-shaped models of Ig domains are shown, with the B and F strand Cys residues at the points of the C (Williams and Barclay, 1988). The first two domains are fused to designate the shared strand. Cys residues are shown in their approximate positions, as are N-glycosylation sites at Asn and Asn. N-Glycosylation sites COOH-terminal to Asn have the sequence Asn-Xaa-Thr and are assumed to be used, based on the sizes of truncated forms of -agglutinin (Wojciechowicz et al., 1993). Another possible N-glycosylation site at Asn is not shown. Only representative O-glycosylations are shown.

There are four cysteine residues in domain III, in the A, B, C`, and F strands. Intradomain disulfide linkages in Ig-like domains often form between cysteines of the B and F strands (Williams and Barclay, 1988). Although Cys and Cys are aligned in positions for the consensus intradomain disulfide bond, Cys in strand A and Cys in strand F form the actual disulfide linkage. The position of the disulfide Cys residues is not as highly conserved in the Ig superfamily as it is in the antibodies themselves. In domain I of myelin-associated glycoprotein, residues in strands B and E of the IgV domain form an intrasheet disulfide linkage (Pedraza et al., 1990). In domain II of CD4, there is a disulfide between strands C and F (Ryu et al., 1990; Wang et al., 1990). Thus, the bond between the A and F strands in domain III of -agglutinin is a new position for intradomain disulfides in the Ig superfamily. These strands are close enough to allow formation of the bond (Lipke et al., 1995).

Cys in strand B and Cys in strand C` of domain III of -agglutinin are free sulfhydryls and can be derivatized under nonreducing conditions. However, they appear not to be exposed to solvent, since they were derivatized only under denaturing conditions (data not shown). A free sulfhydryl is present in at least one other members of the Ig superfamily. CD8 has a single IgV domain with three Cys residues, one of which was in the reduced state in the crystal structure (Leahy et al., 1992). As in -agglutinin, all Cys residues are buried in the interior of the domain.

Glycosylation in -Agglutinin

There is at least one other N-glycosylated residue in -agglutinin. Endo H treatment converts the 21-kDa Arg-C digestion fragment to the 16-kDa fragment, so Asn, Asn, or Asn must be glycosylated. The 5-kDa size difference would accommodate less than 30 carbohydrate residues, the equivalent of a single N-linked chain in yeast (Hames, 1990; Klis, 1994). The glycosylated residue is probably Asn, because it is the only Asn-Xaa-Thr sequence in this part of the molecule, and we have repeatedly failed to obtain the sequence from this residue (peptides T1, DT1, and DS2).

O-Glycosylation is common for cell surface proteins, with O-linked oligosaccharides often in Ser/Thr-rich regions. Many known cell surface O-glycosylated proteins, like low density lipoprotein receptor (Goldstein et al., 1985), decay-accelerating factor (Reddy et al., 1989), the muscle-specific isoform of N-CAM (Walsh et al., 1989), and yeast Gas1p/Gpp1p (Gatti et al., 1994) contain clusters of Ser/Thr enrichment segments in the regions proximal to the membrane. Expression of low density lipoprotein receptor and decay-accelerating factor in mutant cells defective for O-glycosylation result in a rapid cleavage of the binding region from the extracellular surface (Kozarsky et al., 1988; Reddy et al., 1989). In -agglutinin, the region rich in hydroxy amino acids extends from about residue 300 (the F-strand Cys of domain III) to the COOH-terminal signal for GPI anchor addition at approximately residue 627 (Lipke et al. 1989; Kodukula et al., 1993; Wojciechowicz et al., 1993).

-Agglutinin expressed in the presence of tunicamycin, which inhibits N-glycosylation, reacts with ConA, indicating the presence of O-linked mannose residues (Terrance et al., 1987). This binding is not due to reaction with modified GPI anchors, because truncated fragments of -agglutinin lacking the GPI anchor signal also bind ConA (Terrance et al., 1987; Hauser and Tanner, 1989; Wojciechowicz et al., 1993). The pattern of O-glycosylation in -agglutinin indicates that there are multiple sites glycosylated after residue 282, which is at the NH-terminal end of the E strand of domain III. O-Glycosylation is predicted to continue through the Ser/Thr-rich sequence which extends to about residue 620. Six additional Asn-Xaa-Thr sequences in this Ser/Thr-rich region are probably glycosylated based on molecular size of truncated -agglutinin species before and after treatment with endo H (Wojciechowicz et al., 1993). This highly glycosylated region (residues 300-627) would form a ``stalk'' holding the active site out from the wall surface, consistent with electron micrographs (Jentoft, 1990; Cappellaro et al., 1994). Finally, the stalk is predicted to continue to the COOH-terminal GPI anchor, which is processed in vivo to allow linkage to cell wall polysaccharides (Lu et al., 1994, 1995).

A drawing of -agglutinin shows three sequential Ig domains, with N-glycosylation in sites common for such domains (Fig. 10). The binding site includes residues in domain III and at least one other region. The disulfide bonds between domains I and II and between the A and F strands in domain III are unique among Ig domains, and there are two free sulfhydryls in domain III. Following the Ig domains, there is a heavily N- and O-glycosylated stalk sequence, and the COOH-terminal of the protein is initially GPI anchored. Therefore -agglutinin has a structure that recapitulates many of the features of cell adhesion proteins in multicellular eukaryotes.

FOOTNOTES

*

This work was supported by the National Institute for General Medical Science and the Research Centers in Minority Institutions Program of the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§

Current address: Dept. of Pathology, Harvard Medical School, 200 Longwood Ave., Boston MA 02115.

¶

Current address: Dept. of Pharmacology and Toxicology, Dartmouth School of Medicine, Hanover, NH 03755.

**

To whom correspondence should be addressed: Dept. of Biological Sciences, Hunter College, 695 Park Ave., New York, NY 10021. Tel.: 212-772-5235; Fax: 212-772-4073; lipke@genectr.hunter.cuny.edu.

()

The abbreviations used are: GPI, glycosylphosphatidylinositol; ConA, concanavalin A; DTT, dithiothreitol; endo H, endo-N-acetylglucosaminidase H; Ig, immunoglobulin; IgV, immunoglobulin variable domain; P-2007, N-(1-pyrenemethyl)iodoacetamide; PAGE, polyacrylamide gel electrophoresis; TCP, tris-(2-carboxymethyl)phosphine hydrochloride; PGK, phosphoglycerate kinase; HPLC, high performance liquid chromatography.

()

H. De Nobel, P. N. Lipke, and J. Kurjan, submitted for publication.

ACKNOWLEDGEMENTS

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