©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Structure of Saccharomyces cerevisiae -Agglutinin
EVIDENCE FOR A YEAST CELL WALL PROTEIN WITH MULTIPLE IMMUNOGLOBULIN-LIKE DOMAINS WITH ATYPICAL DISULFIDES (*)

(Received for publication, June 1, 1995; and in revised form, August 8, 1995)

Min-Hao Chen (1)(§) Zheng-Ming Shen (1) Stephen Bobin (1)(¶) Peter C. Kahn (2) Peter N. Lipke (1)(**)

From the  (1)Department of Biological Sciences and The Institute for Biomolecular Structure and Function, Hunter College of the City University of New York, New York 10021 and the (2)Department of Biochemistry and Microbiology, Cook College, Rutgers University, New Brunswick, New Jersey 08903

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

alpha-Agglutinin of Saccharomyces cerevisiae is a cell wall-associated protein that mediates cell interaction in mating. Although the mature protein includes about 610 residues, the NH(2)-terminal half of the protein is sufficient for binding to its ligand a-agglutinin. alpha-Agglutinin, a fully active fragment of the protein, has been purified and analyzed. Circular dichroism spectroscopy, together with sequence alignments, suggest that alpha-agglutinin consists of three immunoglobulin variable-like domains: domain I, residues 20-104; domain II, residues 105-199; and domain III, residues 200-326. Peptide sequencing data established the arrangement of the disulfide bonds in alpha-agglutinin. Cys is disulfide-bonded to Cys, forming an interdomain bond between domains I and II. Cys is bonded to Cys, in an atypical intradomain disulfide bond between the A and F strands of domain III. Cys and Cys have free sulfhydryls. Sequencing also showed that at least two of three potential N-glycosylation sites with sequence Asn-Xaa-Thr are glycosylated. At least one of three Asn-Xaa-Ser sequences is not glycosylated. No residues NH(2)-terminal to Ser were O-glycosylated, whereas Ser, and all hydroxy amino acid residues COOH-terminal to this position were modified. Therefore O-glycosylated Ser and Thr residues cluster in the COOH-terminal region of domain III, and the O-glycosylation continues into a Ser/Thr-rich sequence that extends from domain III to the COOH-terminal of the full-length protein.


INTRODUCTION

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 alpha-agglutinin is a highly glycosylated cell wall-anchored protein that is constitutively expressed on cells of the alpha 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 alpha-agglutinin gene, AGalpha1, encodes a single polypeptide of 650 amino acids, including an NH(2)-terminal secretion signal (residues 1-19) and a COOH-terminal glycosylphosphatidylinositol (GPI) (^1)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(2)-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 alpha-agglutinin sequence. The open reading frame of the Agalpha1 gene is shown. The NH(2)-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(2)-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 beta-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. (^2)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 alpha-agglutinin (Wojciechowicz et al., 1993; Lu et al., 1994).

We have now produced a 332-residue active fragment, alpha-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 alpha-agglutinin.


EXPERIMENTAL PROCEDURES

Chemicals and Reagents

All chemicals were from Sigma, unless otherwise stated, and of appropriate purity. Nitrocellulose membranes were from Schleicher & Schuell. Reagents for gel electrophoresis were from Kodak Scientific Imaging Systems. Protein standards and Bio-Gel P-60 were purchased from Bio-Rad. Reagents for polymerase chain reactions were obtained from Perkin-Elmer, and restriction enzymes were from New England Biolabs or U. S. Biochemical Corp. Endoprotease Arg-C, sequencing grade Staphylococcus aureus V8, hydrophilic bead-bound trypsin, and endoprotease Asn-N were from Boehringer Mannheim. The cysteine-specific reagent P-2007 (N-(1-pyrenemethyl)iodoacetamide) and reducing reagent TCEP (tris-(2-carboxyethyl)phosphine hydrochloride) were from Molecular Probes. Immobilon-AV membranes were purchased from Millipore.

Yeast Strains and Expression Vector

The agalpha1-3 mutant (Lalpha21) (Lipke et al., 1989), which is isogenic to W303-1B (MATalpha ade2-1 his3-11, 15 leu2-3, 112 trp1-1 ura3-2 can1-100), was used to express the alpha-agglutinin construct pPGK-AGalpha1. Bioassays utilized tester strain X2180-1A (MATalpha SUC2 mal mel gal2 CUP1) and X2180-1B (MATalpha SUC2 mal mel gal2 CUP1) (Terrance and Lipke, 1981). The expression vector, YEp-PGK, containing the pBR322-derived Amp^R and Ori^E, the yeast URA3 gene, and 2-µm replication origin, allowed the cloning of the AGalpha1 fragment between the constitutive phosphoglycerate kinase (PGK) promoter and terminator (Kang et al., 1990). Plasmids were propagated in Escherichia coli strain HB101.

Construction of pPGK-AGalpha1

Two single-stranded oligonucleotides were synthesized to use as primers for the construction of pPGK-AGalpha1. AGalpha5`-H3`, TTC GCC AAG CTT TTC AAA ATG TTC ACT TTT CTC, and AGalphaM-H3`, AAA TGG AAG CTT TGG ATT ACG CAC TAG TGT TTA TAC TTG T, contain HindIII sites (underlined nucleotides) outside the open reading frame. The 3` end primer included a stop codon (nucleotides with double underline) corresponding to Tyr in the deduced alpha-agglutinin protein sequence. The DNA fragment encoding alpha-agglutinin was amplified using the AGalpha1-containing plasmid pH27 (Lipke et al., 1989) as template in a polymerase chain reaction. The polymerase chain reaction product contained the open reading frame of AGalpha1 from nucleotides 1 to 1053 and included the sequence encoding the secretion signal. The purified polymerase chain reaction product was cloned into the HindIII site of the expression vector YEp-PGK. The orientation of the insert was checked by restriction mapping with EcoRI, HindIII, and BamHI, and the sequence of the insert in pPGK-AGalpha1 was verified by DNA sequencing.

Overexpression and Purification of alpha-Agglutininfrom Culture Supernatant

pPGK-AGalpha1, encoding alpha-agglutinin, was introduced into the agalpha1 mutant Lalpha21. Transformants were grown to stationary phase in 1-liter cultures of synthetic uracil-less medium overnight at room temperature. The cells were centrifuged, and the culture supernatant was concentrated 10-fold through a Millipore filtration apparatus equipped with a membrane having a 100-kDa molecular weight cutoff. Aliquots of concentrated supernatant (50 ml) were dialyzed par overnight against 4 liters of 10 mM sodium acetate buffer, pH 5.5, at 4 °C. The dialyzed material was partially purified by chromatography on a DEAE-Sephadex column (120-ml bed volume) which was previously equilibrated with 10 mM sodium acetate, pH 5.5. The column was washed with the same buffer and eluted with 300 mM sodium chloride, 10 mM sodium acetate, pH 5.5, in 3-ml fractions. The alpha-agglutinin content of each eluted fraction was determined by assaying for agglutinin activity (Terrance and Lipke, 1981). Fractions containing activity were pooled for further purification.

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 alpha-agglutinin. The de-N-glycosylated alpha-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

Rabbit polyclonal antisera against alpha-agglutinin were raised by injection of purified deglycosylated alpha-agglutinin. Immunoblots were performed as described previously (Harlow and Lane, 1988; Wojciechowicz and Lipke, 1989). Briefly, after overnight transfer of proteins from SDS gels to nitrocellulose membranes, the membranes were blocked with 3% gelatin in phosphate-buffered saline and then incubated in the same buffer with 1% gelatin, 0.1% Tween 20, and a 1:1000 dilution of antibody that had been adsorbed with heat-killed a cells. A second incubation followed with a 1:1000 dilution of peroxidase-conjugated goat anti-rabbit-IgG antibody (Sigma) in the same buffer. The blots were stained by peroxidase-mediated reaction of 4-chloro-1-naphthol with hydrogen peroxide.

CD and Structure Analysis

Cuvettes of 0.1-cm path length were used for far-UV spectra, with typically five spectra being accumulated, averaged, and base line-corrected on an AVIV CD spectrometer model 60 DS (Lakewood, NJ) interfaced to an IBM personal computer. All spectra were acquired at 25 °C. For conversion to mean residue ellipticity, a mean residue weight of 111.77 was used. The program PROSEC (Yang et al., 1986) was used to analyze secondary structure distribution from recorded CD data. Smoothing was by the Gram method.

Endoprotease Digestions

Proteolytic digestions were initially conducted on heat-denatured alpha-agglutinin in the presence of 10% acetonitrile for 18 h at 25 °C, according to the manufacturer' suggested protocols. The ratio of protease to substrate was 1:25 for trypsin and S. aureus V8, 1:100 for endoprotease Arg-C, and 1:5 for endoprotease Asn-N, respectively. Some trypsin and S. aureus V8 digestions were performed on nondenatured alpha-agglutinin. Endoprotease Arg-C digestions were performed in 0.1 M NH(4)HCO(3) buffer, pH 7.8. S. aureus V8 digestions were performed in 0.1 M sodium phosphate buffer, pH 7.8. Under these conditions, S. aureus V8 cleaves at the COOH-terminal side of both Glu and Asp, with some cleavage at Asn and Gln residues (Drapeau, 1978). Trypsin digestions were performed in 0.1 M Tris buffer, pH 8.0. HPLC purified tryptic peptides were lyophilized to remove acetonitrile and trifluoroacetic acid, prior to digestion with endoprotease Asp-N digestion in 100 mM Tris buffer, pH 7.0.

HPLC

Peptide mixtures derived from digests and reduced digests were fractionated by reversed phase HPLC on an Applied Biosystems instrument using a fully end-capped Microbore Vydac C18 (3 cm times 3 mm inner diameter, 5 µm) with a Brownlee RP-300 guard column. Solvent A was 0.1% (v/v) aqueous trifluoroacetic acid and solvent B was 90% acetonitrile in water (v/v) containing 0.1% trifluoroacetic acid (v/v). The solvent elution rate was at 50 µl/min. The column effluent was monitored by absorbance at 220 nm, and peptide peaks were collected manually. For most tryptic digestions, products were fractionated with a linear gradient from 0 to 60% solvent B in 180 min. For identification of cysteine-specific labeled tryptic peptides, the gradient was programmed linearly from 0% solvent B to 100% solvent B in 200 min. For S. aureus V8 digestion, all peptides were fractionated by a linear solvent gradient from 0 to 45% solvent B in 180 min.

Cysteine-specific Labeling of Tryptic Peptides of alpha-Agglutinin

The cysteine-specific reagent, P-2007, was used to label free sulfhydryl groups. The reaction was performed overnight at room temperature in 80% dimethyl formamide in 0.1 M phosphate buffer, pH 7.2. In some cases, the digestion mixtures were treated with the reducing reagent TCEP, 7 mM, before labeling. Because TCEP does not react with P-2007, a simplified procedure was used, and the alkylation was carried out in the presence of the reducing reagent. Additives and trypsin beads were removed on a Bio-Gel P-2 spin column after labeling. The labeled mixture was separated on a microbore C-18 HPLC column, and P-2007-labeled peptides were detected at 341 nm.

NH(2)-terminal Sequencing of Peptides

Peptides were sequenced by automated Edman degradation in a gas-phase sequenator (model 470A, Applied Biosystems Inc.). The resulting phenylthiohydantion-derivatized amino acid residues were separated on a Vydac C18 column using a 120A phenylthiohydantion analyzer (Applied Biosystems Inc.). Individual amino acid residues were identified and quantitated by comparison with standards. Peptides resolved and sequenced are summarized in Fig. 8.


Figure 8: Summary of sequenced alpha-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 alpha-AgglutininPeptides

Each fraction from the reversed phase column was vacuum-evaporated to dryness and resuspended in 30 µl of 0.5 M sodium phosphate buffer, pH 8.0, containing 0.1% SDS (w/v). Peptide samples (3 times 1 µl) were spotted onto an Immobilon-AV membrane. The membrane was air-dried and incubated for 30 min in 10 mM Tris-HCl buffer, pH 7.5, containing 0.15 M sodium chloride and 0.1% Tween 20 (TTBS) and then blocked in a 3-h incubation in with fresh 10% ethanolamine (v/v) in 1 M sodium bicarbonate buffer, pH 9.5. After blocking, the membranes were incubated for 1 h with 0.5 µg/ml concanavalin A (ConA)-conjugated peroxidase in TTBS. After washing three times in 10 mM Tris-HCl buffer, pH 7.5, containing 0.15 M NaCl, the membranes were stained with 4-chloro-1-naphthol and hydrogen peroxide (Canas et al., 1993).

Other Methods

SDS-polyacrylamide gel electrophoresis was carried out according to the method of Laemmli(1970), using 12 and 15% gels. Proteins were visualized by staining with Coomassie Blue or a Silver Staining Plus kit (Bio-Rad). Protein concentrations were determined by bicinchoninic protein assay method (Pierce) using bovine serum albumin, fraction V as standard.


RESULTS

Expression and Secretion of alpha-Agglutinin

The plasmid pPGK-AGalpha1 encodes a 351-residue form of alpha-agglutinin that lacks the COOH-terminal sequences which anchor alpha-agglutinin to the cell wall (Wojciechowicz et al., 1993), and therefore the product, alpha-agglutinin, is secreted into the culture medium after cleavage of the 19-residue secretion signal. An agalpha1 mutant harboring this plasmid secreted 4.5 times 10^4 units (about 1 mg) of alpha-agglutinin/liter (Terrance et al., 1987; Wojciechowicz et al., 1993). alpha-Agglutinin in crude culture supernatants was identified by immunoblots before and after endoglycosidase H treatment (Fig. 2). The fully glycosylated protein had an apparent molecular size of 110 kDa. After removal of N-linked carbohydrates with endo H, the molecular size of alpha-agglutinin was reduced to 45 kDa. In some preparations, the protein was present as a doublet (Fig. 3), due to incomplete removal of N-linked glycan at one site (data not shown). The mobility of the deglycosylated alpha-agglutinin decreased after treatment with DTT (Fig. 2). This decrease implies an increase in the Stokes radius caused by reduction of disulfide bonds.


Figure 2: Immunoblot of alpha-agglutinin from culture supernatant. Supernatant from a culture of Lalpha21 [pPGK-AGalpha1] (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 alpha-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 alpha-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 alpha-agglutinin from a Bio-Gel P-60 column gave purified alpha-agglutinin with an apparent molecular size of 45 kDa for the smaller species on SDS gels (Fig. 3). The deduced M(r) of alpha-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 alpha-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 alpha-Agglutinin

When purified alpha-agglutinin was digested with mouse endoprotease Arg-C from mouse submaxillary glands, proteolytic fragments of 31, 21, and 16 kDa were generated (Fig. 4).


Figure 4: SDS-PAGE analysis of endoprotease Arg-C-digested alpha-agglutinin. Samples of endoprotease Arg-C-digested alpha-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(2)-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(2)-terminal sequence as mature alpha-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(2)-terminal alpha-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 alpha-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 alpha-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 alpha-agglutinin incubated without protease. Therefore, hydrolysis of alpha-agglutinin at Lys was endoprotease Arg-C specific and not due to proteolytic activity in the alpha-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 alpha-Agglutinin

To examine whether any of the endoprotease Arg-C digested fragments retained agglutination activity, protease-treated alpha-agglutinin was reconstituted with sodium acetate buffer to pH 5.5 and assayed for activity. This material had no measurable agglutination activity at concentrations up to 6.7 µg/ml, whereas native alpha-agglutinin was active at 3.3 ng/ml. Therefore the agglutination activity was less than 2 times 10 that of intact alpha-agglutinin. Similarly, the 31-kDa alpha-agglutinin fragment purified on a Bio-Gel P-30 column had less than 10 of the binding activity of alpha-agglutinin (data not shown).

CD of Native alpha-Agglutinin

alpha-Agglutinin has been proposed to consist of three Ig-like domains, which would consist of predominantly antiparallel beta-sheets along with associated turns and loops, but little or no alpha-helix content (Williams and Barclay, 1988; Wojciechowicz et al., 1993). The CD spectrum of alpha-agglutinin (Fig. 5) showed a typical beta-sheet structure profile, with a negative band at 217 nm (Brahms and Brahms, 1980). The absence of the intense negative peaks at either 208 or 222 nm, which are the characteristic of alpha-helix, indicated very little alpha-helix content in alpha-agglutinin. Quantitative analysis of the CD spectrum of alpha-agglutinin indicated the presence of 6.8% alpha-helix, 69.4% beta-sheet, 13.2% turns, and 10.5% random structure. This high beta-sheet content suggests the presence of antiparallel beta-sheet structures, consistent with Ig domains.


Figure 5: Far-UV CD spectra of alpha-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) alpha-agglutinin and endoprotease Arg-C-digested alpha-agglutinin (dashed line).



CD of alpha-AgglutininDigested with Endoprotease Arg-C

Because alpha-agglutinin is inactivated by endoprotease Arg-C cleavage at Lys, the effect of the digestion on the structure of alpha-agglutinin fragments was examined. The digestion product showed substantial reduction in beta-sheet content when spectra were taken at pH 7.8 (Fig. 5). However, after reconstitution at pH 5.5 for 30 min, the CD spectrum of the digest was very similar to that of native alpha-agglutinin, in both the negative peak position at 217 nm and the corresponding peak width (data not shown). Quantitative analysis of the CD spectrum revealed that the secondary structural profile was similar to native alpha-agglutinin, with 68.8% beta-sheet, and a slightly higher aperiodic structure content. This CD profile indicated that the single site digestion at Lys of alpha-agglutinin did not substantially alter the secondary structure of the protein fragments. Therefore, the inactivation of the binding activity is not due to gross structural change during the Arg-C digestion.

Disulfides in Endoprotease Arg-C-digested alpha-Agglutinin

The products of endoprotease Arg-C cleavage of alpha-agglutinin were separable in the absence of reducing agents (Fig. 4), showing that there is no disulfide linkage between them. Both the 21- and the 31-kDa fragments showed lower mobility on SDS-PAGE after DTT treatment, suggesting that each fragment contained one or more internal disulfide bonds. Based on the deduced amino acid sequence, the 21-kDa fragment contained Cys and Cys, implying that these two residues form a disulfide bond. The 31-kDa fragment, alpha-agglutinin, contained four Cys residues (Cys, Cys, Cys, and Cys). Therefore, the disulfide bonds in this fragment could not be determined from the endoprotease Arg-C data.

Identification of Disulfide Bonds

Identification of these disulfide bonds was accomplished by sequencing of tryptic and S. aureus V8 peptides that had different HPLC retention times in the presence and absence of DTT. Free sulfhydryls were identified in peptides that were not affected by DTT and confirmed by labeling with the iodoacetamide derivative P-2007.

alpha-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 alpha-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 alpha-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 alpha-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

Tryptic peptide peak T4 from nonreduced alpha-agglutinin and peak DT4 from reduced alpha-agglutinin had a retention time of 155 min (Fig. 6) and yielded the same sequence containing Cys ( Table 4and Table 5). There is no tryptic site between Cys and Cys (Table 3); therefore this peptide should contain both cysteines. This peptide does not appear to include a disulfide bond, because the retention time was not altered by reduction. In support of the presence of free sulfhydryls in this region, a peptide, S4, including a single Cys residue (Cys) was obtained and sequenced from S. aureus V8 digestion under both nonreduced and reduced conditions (Table 5). Therefore, Cys has a free sulfhydryl.



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

We have sequenced all recovered tryptic and S. aureus V8 peptides from alpha-agglutinin, resulting in a peptide sequence that is about 76% complete, and including three of six potential N-glycosylation sequences and 52 of 74 Ser and Thr residues (Fig. 8). Glycosylated Ser or Thr residues are not detected by the sequencer; therefore, peptide sequencing provides an indirect method to identify O-linked glycosylation sites. Absence of a signal for Thr and Ser was interpreted to indicate glycosylation when the expected residues were observed at levels of 20 pmol or greater in the cycles immediately preceding missing Ser or Thr residues. Table 6summarizes the results from sequencing of S. aureus V8 and tryptic alpha-agglutinin peptides from two or more independent peptide sequences. A total of four S. aureus V8 peptides and two tryptic peptides contained modified Ser and Thr residues.



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 alpha-agglutinin were modified. All other sequenced Ser and Thr residues were observed as expected (Fig. 8).

Confirmation of O-Glycans with ConA

O-Linked carbohydrates in yeast interact with ConA, because they consist of one to five alpha-linked mannose residues (Klis, 1994). To examine whether O-linked glycosylations were responsible for the masking of the undetected Ser and Thr residues, peroxidase-conjugated ConA was used to probe peptides from the nonreduced tryptic digest. Dot blot analysis of tryptic fractions of HPLC fractions of nonreduced digest showed that five peptides reacted positively with ConA (data not shown). These peptides (fractions 4, 5, 24, 25, and 26 of Fig. 6A) correlated with fragments containing modified Ser and Thr residues (Table 6). Because the dot blot experiment does not determine which Ser or Thr residues within a peptide were glycosylated, we cannot definitively conclude that O-glycosylation accounts for all of the modification of Ser or Thr residues in these peptides, but it must account for some.

Identification of N-Linked Glycosylation Sites in alpha-Agglutinin

Endo H cleaves between the two GlcNAc residues of N-linked oligosaccharides, leaving one GlcNAc attached to Asn. The modified Asn residue is not detectable by the sequencer and therefore provides an indirect assay for N-glycosylation. There are six potential N-linked glycosylation sites (Asn-Xaa-Ser/Thr) in alpha-agglutinin. Asn-Asp-Thr and Asn-Thr-Thr were N-glycosylated, whereas Asn-Thr-Ser was not N-glycosylated in (Table 6: tryptic peptide 326-351). Other potential N-glycosylation sequences were in regions that were not successfully sequenced.


DISCUSSION

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

Domains of alpha-Agglutinin

IgV domains consist of nine antiparallel strands, (A, B, C, C`, C", D, E, F, and G) having strongly conserved residues in the B, C, D, and F strands (Williams and Barclay, 1988). For domain III there are highly significant sequence similarities to an IgV consensus sequence, especially in the B, C, and F strands (Wojciechowicz et al., 1993). The complete domain, including A and G strands, extends the alignment to residues 200-326 (Fig. 9). A three-dimensional model of domain III based on homology to IgV domains has been constructed that accommodates the disulfide bond between Cys and Cys, positions of glycosylated residues and proteolytic sites, CD spectra, and site-specific mutagenesis results (Lipke et al., 1995).^2 Thus, an IgV-like structure for domain III can accommodate all available data.


Figure 9: Alignment of three domains of alpha-agglutinin with each other and with a consensus sequence for IgV domains (Williams and Barclay, 1988). The positions of the beta-strands in the consensus sequence are shown. The alignment is based on secondary structure prediction and alignment within prospective beta-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(2)-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(2) 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(2)-terminal residues of alpha-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 alpha-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 alpha-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 alpha-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 alpha-agglutinin. In the three proposed alpha-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 alpha-Agglutinin in the Ig Superfamily

The CD spectrum of alpha-agglutinin was similar to those of other members of the Ig superfamily, showing little or no alpha-helix and a predominance of beta-sheet. The magnitude of the negative peak at 217 nm characteristic of beta-sheet was greater in alpha-agglutinin than in the spectrum of Igs themselves, but was in the range of that for many other members of the Ig superfamily (Cathou and Dorrington, 1975; Jefferis et al., 1978; Killeen et al., 1988). The CD profile of alpha-agglutinin is similar to those of MRC OX-45, CD4, Thy-1, and CD2 (Campbell et al., 1979; Killeen et al., 1988; Chamow et al., 1990; Recny et al., 1990). The mean residue ellipticity at 217 nm for alpha-agglutinin, Thy-1, and CD2 are -4.68 x 10^3, -4.8 times 10^3, and -6.6 times 10^3 degreesbulletcm^2bulletdmol, respectively. The high beta-sheet content of alpha-agglutinin is also close to that of silk fibroin (Demura and Asakura, 1991) and human plasma fibronectin (Oesterlund, 1988), both of which are mostly antiparallel beta-sheet structures (65 and 79%, respectively), and may be close to the maximum possible beta-sheet content. Such a high beta-sheet content can only be accommodated in globular proteins by antiparallel structures. Therefore, the beta-sheet content of alpha-agglutinin (70%) is among the highest for known proteins with essentially pure antiparallel beta-sheet structures. The unusually high content of antiparallel beta-sheet also implies the presence of antiparallel beta-sheet structure throughout the molecule and is therefore consistent with the three-domain alignment. It is worth noting that, even if domain III were composed of pure antiparallel beta-sheet structure (100% sheet), domain I and II would still have a beta-sheet content of at least 50% to yield an overall beta-sheet content of 70% in alpha-agglutinin. Therefore, beta-sheet is the predominant structure in all of the domains.

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 alpha-agglutinin fragment nor the unpurified Arg-C digest of alpha-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 alpha-Agglutinin

Cys and Cys form an interdomain disulfide bond between the proposed COOH terminus of domain I and the NH(2) terminus of domain II (Fig. 8Fig. 9Fig. 10). Interdomain disulfides are known in other members of the Ig superfamily, including the lymphoid differentiation antigen CD33 (Simmons and Seed, 1988), the B cell adhesion molecule CD22 (Stamenkovic and Seed, 1990) and the myelin-associated glycoprotein (Pedraza et al., 1990), but alpha-agglutinin is unique in the position of the bond between the F and B strands on sequential domains.


Figure 10: Structure of alpha-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 alpha-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 alpha-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 alpha-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. CD8alpha 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 alpha-agglutinin, all Cys residues are buried in the interior of the domain.

Glycosylation in alpha-Agglutinin

alpha-Agglutinin is both N- and O-glycosylated (Terrance et al., 1987; Wojciechowicz et al., 1993; Lu et al., 1994). Our N-glycosylation results conform to the finding that Asn-Xaa-Thr sequences are preferred over Asn-Xaa-Ser as N-glycosylation sites in yeast (Moehle et al. 1987; however, see Riederer and Hinnen(1991)), in that of the three sequenced sites, the two Asn-Xaa-Thr sequences were glycosylated and the Asn-Xaa-Ser sequence was not. The sites of N-glycosylation, between beta-strands C and C` and between strands F and G of domain III, are common in members of the Ig superfamily (Barclay et al., 1993; Dwek et al., 1993).

There is at least one other N-glycosylated residue in alpha-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 alpha-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).

alpha-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 alpha-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 alpha-agglutinin indicates that there are multiple sites glycosylated after residue 282, which is at the NH(2)-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 alpha-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 alpha-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 alpha-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.

(^1)
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.

(^2)
H. De Nobel, P. N. Lipke, and J. Kurjan, submitted for publication.


ACKNOWLEDGEMENTS

We thank Janet Kurjan and Joseph Krakow for helpful comments on the manuscript. We are grateful to Hans de Nobel for helpful comments and for sequencing of the pAGalpha120-351 insert.


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