The complete cDNA and amino acid sequence of bovine fetuin. Its homology with alpha 2HS glycoprotein and relation to other members of the cystatin superfamily.

The cDNA sequence encoding the bovine fetal protein fetuin is reported. The deduced amino acid sequence is identical with that obtained from amino acid sequencing. The protein is a single chain preceded by a signal sequence. The three N-linked glycosylation sites have been determined. The sequence of fetuin shows over 70% similarity to human alpha 2HS glycoprotein. All of the cysteine residues are conserved in both proteins, suggesting that fetuin has the same arrangement of disulfide loops as alpha 2HS glycoprotein and may also be a member of the cystatin family. Southern blot analysis indicates that a single gene codes for fetuin. No evidence for a separate gene for a bovine alpha 2HS glycoprotein was obtained; thus, fetuin in cattle and alpha 2HS glycoprotein in the human are equivalent proteins.     

Early cortical plate specific glycoprotein in a marsupial species belongs to the same family as fetuin and alpha 2HS glycoprotein

Two related glycoproteins, fetuin in species of the order Artiodactyla (cattle, sheep, pig) and alpha 2HS glycoprotein in the human [(1987) Cell Tissue Res. 248, 33-41] have a very specific distribution in the developing brain. We have isolated and determined the first 15 N-terminal residues of a similarly distributed glycoprotein in the developing brain of the tammar wallaby (Macropus eugenii). The degree of homology is the same between wallaby glycoprotein and alpha 2HS glycoprotein as between fetuin and alpha 2HS glycoprotein (46%). Antibodies made to synthetic peptides of fetuin were used to identify the wallaby glycoprotein. A polyclonal antibody to the purified glycoprotein was used for immunocytochemical identification of brain cells positive for this protein.     

Fetuin--an old friend revisited.

Bovine fetuin, the first fetal protein to be described, has recently been shown to be a species homologue of a well known human plasma protein--alpha 2HS glycoprotein (alpha 2HS). The fetuins are now known to be members of the cystatin superfamily. The structural properties of the six fetuins that have been fully sequenced are compared. Despite the structural homology of these proteins, their described properties in the literature make them appear to be quite different. The diverse in vitro properties claimed for fetuin/alpha 2HS are reviewed. In vivo, fetuins are involved in the acute phase response. In development, in all species studied so far, fetuins are present in a specific cell population that forms the developing neocortex. The possible functional significance of this distribution is discussed.     

The nucleotide and deduced amino acid structures of sheep and pig fetuin. Common structural features of the mammalian fetuin family.

This study was initiated to gain further insight into the structural features of the mammalian fetuin family. The cDNA structures of sheep and pig fetuin were determined. The cDNA insert encoding sheep (pig) fetuin comprised 1550 (1470) nucleotides, including 54 (46) nucleotides encoding a signal peptide of 18 (15) residues and 1038 (1041) nucleotides encoding the 346 (347) amino acids of the mature plasma protein. The predicted amino-terminal sequence of the mature pig fetuin was confirmed by the amino-terminal sequence of the purified protein. However, two alternative sheep amino-terminal sequences were found in fetuin purified from the plasma of a single sheep fetus; the minor product was the one predicted by comparison with other fetuin sequences while the major product was two amino acids longer. Comparison of the deduced amino acid sequences of sheep and pig fetuin showed an extensive sequence identity between them (75%) and with other proteins of the mammalian fetuin family, i.e. human alpha 2-HS glycoprotein, and bovine and rat fetuins. Twelve cysteine residues were found at invariant positions in all fetuin sequences, suggesting strongly that the arrangement of disulphide bridges identified in human alpha 2-HS glycoprotein is common to the members of the family. Further sequence comparisons revealed that the structures of mammalian fetuins are organised in three domains: two cystatin-like domains (D1 and D2) and a complex carboxyl-terminal domain (D3). The proposed three-domain structure of the protein is reflected in the organisation of the rat fetuin structural gene which has recently been published.     

Specific proteolysis of native alanine racemases from Salmonella typhimurium: identification of the cleavage site and characterization of the clipped two-domain proteins

Native DadB and Alr alanine racemases (Mr 39,000) from Salmonella typhimurium are proteolyzed at homologous positions by alpha-chymotrypsin, trypsin, and subtilisin to generate in all cases two nonoverlapping polypeptides of Mr 28,000 and 11,000. Under nondenaturing conditions, chymotryptic digest results in an associated form of the two fragments which possesses 3% of the original catalytic activity, incorporates 0.76 equiv of the mechanism-based inactivator beta-chloro-[14C]-D-alanine [Badet, B., Roise, D., & Walsh, C. T. (1984) Biochemistry 23, 5188], and exhibits a UV circular dichroism profile identical with that of native enzyme. Protein sequence analysis of the denatured chymotryptic fragments indicates the presence of a tetrapeptide interdomain hinge (DadB, residues 254-257; Alr, residues 256-259) that is attacked at both ends during proteolysis. Under the previously employed digest conditions, NaB3H4-reduced DadB holoenzyme is resistant to alpha-chymotrypsin and trypsin and is labile only toward subtilisin. These data suggest that the hinge structure is essential for a catalytically efficient enzyme species and is sensitive to active site geometry. The sequence at the hinge region is also conserved in alanine racemases from Gram-positive bacteria.     

Monoclonal antibodies to two glycoproteins of herpes simplex virus type 2.

Monoclonal antibodies to herpes simplex virus type 2 were found to precipitate different numbers of radiolabeled polypeptides from lysates of virus-infected cells. Antibodies directed against two viral glycoproteins were characterized. Antibodies from hybridoma 17 alpha A2 precipitated a 60,000-molecular-weight polypeptide which chased into a 66,000- and 79,000-molecular-weight polypeptide. All three polypeptides labeled in the presence of [3H]glucosamine and had similar tryptic digest maps. The 60,000-molecular-weight polypeptide also chased into a 31,000-molecular-weight species which did not label with [3H]glucosamine. Antibodies from hybridoma 17 beta C2 precipitated a 50,000-molecular-weight polypeptide which chased into a 56,000- and 80,000-molecular weight polypeptide. These polypeptides also shared a similar tryptic digest map and labeled with [3H]glucosamine. Both monoclonal antibodies were herpes simplex virus type 2 specific. The viral proteins precipitated by 17 alpha A2 antibodies had characteristics similar to those reported for glycoprotein E, whereas the proteins precipitated by 17 beta C2 antibodies appeared to represent a glycoprotein not previously described. This glycoprotein should be tentatively designated glycoprotein F.     

Multiple enzymatic digestion for enhanced sequence coverage of proteins in complex proteomic mixtures using capillary LC with ion trap MS/MS

This study uses multiple enzyme digests to increase the sequence coverage of proteins identified by the shotgun sequencing approach to proteomic analysis. The enzymes used were trypsin, Lys-C, and Asp-N, which cleave at arginine and lysine residues, lysine, and aspartic acid residues, respectively. This approach was evaluated with the glycoprotein, tissue plasminogen activator, t-PA and gave enhanced sequence coverage, compared with a single enzymatic digest. The approach was then evaluated with a complex proteomic sample, namely plasma. It was found that trypsin and Lys-C were able to detect overlapping but distinct sets of proteins and a digital recombination of the data gave a significant increase in both the number of protein identifications as well as an increase in the number of peptides identified per protein (which improves the certainty of the assignment).     

Site-directed mutagenesis of a soluble recombinant fragment of platelet glycoprotein Ib alpha demonstrating negatively charged residues involved in von Willebrand factor binding.

We have expressed in mammalian cells a fragment (residues 1-302) of the alpha chain of platelet glycoprotein (GP) Ib containing the von Willebrand factor- (vWF) binding site. The secreted soluble protein had an apparent molecular mass of 45 kDa and reacted with conformation-dependent monoclonal antibodies that bind only to native GP Ib, thus demonstrating its proper folding. After insolubilization on nitrocellulose membrane, the recombinant GP Ib alpha fragment bound soluble vWF in the presence of ristocetin or botrocetin with a dissociation constant similar to that exhibited by GP Ib.IX complex on platelets. Moreover, the interaction was blocked by anti-GP Ib monoclonal antibodies known to inhibit vWF binding to platelets. The sequence of GP Ib alpha between residues 269-287 has a strong net negative charge due to the presence of 10 glutamic or aspartic acid residues; 5 of these are contained in the sequence of a synthetic peptide (residues 251-279) previously shown to inhibit vWF-platelet interaction. In order to evaluate the possible functional role of these acidic residues, we employed site-directed mutagenesis to express two mutant GP Ib alpha fragments containing asparagine or glutamine instead of aspartic or glutamic acid, respectively. Mutant 1, with substitutions between residues 251-279, failed to bind vWF whether in the presence of ristocetin or botrocetin; in contrast, vWF binding to Mutant 2, with substitutions between residues 280-302, was nearly normal in the presence of ristocetin, but markedly decreased in the presence of botrocetin. Thus, mammalian cells transfected with a truncated cDNA sequence encoding the amino-terminal domain of GP Ib alpha synthesize a fully functional vWF-binding site; acidic residues in the sequence 252-287 are essential for normal function.     

Localization of discontinuous epitopes of herpes simplex virus glycoprotein D: use of a nondenaturing ("native" gel) system of polyacrylamide gel electrophoresis coupled with Western blotting

Previously, a panel of monoclonal antibodies (MCAb) was used to define specific epitopes of herpes simplex virus glycoprotein D (gD) (R. J. Eisenberg et al., J. Virol. 53:634-644, 1985). Three groups of antibodies recognized continuous epitopes; group VII reacted with residues 11 to 19 of the mature protein (residues 36 to 44 of the predicted sequence), group II reacted with residues 272 to 279, and group V reacted with residues 340 to 356. Four additional antibody groups recognized discontinuous epitopes of gD, since their reactivity was lost when the glycoprotein was denatured by reduction and alkylation. Our goal in this study was to localize more precisely the discontinuous epitopes of gD. Using a nondenaturing system of polyacrylamide gel electrophoresis ("native" gel electrophoresis) coupled to Western blotting, we analyzed the antigenic activity of truncated forms of gD. These fragments were generated either by recombinant DNA methods or by cleavage of purified native gD-1 (gD obtained from herpes simplex virus type 1) and gD-2 (gD obtained from herpes simplex virus type 2) with Staphylococcus aureus protease V8. Antibodies in groups III, IV, and VI recognized three truncated forms of gD-1 produced by recombinant DNA methods, residues 1 to 287, 1 to 275, and 1 to 233. Antibodies in group I recognized the two larger forms but did not react with the gD-1 fragment of residues 1 to 233. On the basis of these and previous results, we concluded that a protion of epitope I was located within residues 233 to 259 and that epitopes III, IV, and VI were upstream of residue 233. Antibodies to continuous epitopes identified protease V8 fragments of gD-1 and gD-2 that contained portions of either the amino or carboxy regions of the proteins. None of the V8 fragments, including a 34K polypeptide containing residues 227 to 369, reacted with group I antibodies. This result indicated that a second portion of epitope I was located upstream of residue 227. Two amino-terminal fragments of gD-1, 33K and 30K, reacted with group III, IV, and VI antibodies. A 33K fragment of gD-2 reacted with group III antibodies. Based on their size and reactivity with endo-beta-N-acetylglycosaminidase F, we hypothesized that the 33K and 30K molecules represented residues 1 to 226 and 1 to 182 of gD-1, respectively. These results suggest that epitopes III, IV, and VI are located within the first 182 residues of gD.(ABSTRACT TRUNCATED AT 400 WORDS)     

Simplified elimination of rabbit anti-rat acute phase protein IgG that shows cross reactivity with albumin

Antibody preparations against rat acute phase proteins were tested for cross reactivity with other serum proteins, including rat albumin. Rabbit anti-rat a alpha1-acid glycoprotein and ceruloplasmin IgG purified on protein A-Sepharose did not show any cross reactivity with rat albumin, hemoglobin or transferrin. Rabbit anti-rat haptoglobin and -macroglobulin IgG purified on protein A-Sepharose showed a 39% and 30% cross reactivity with rat albumin and a 20% and 19% cross reactivity with rat hemoglobin. Because these proteins in whole serum were not adsorbed on Cibacron Blue F3-GA Sepharose, the albumin would be adsorbed on Cibacron Blue F3-GA Sepharose by the use of whole rat serum. Rabbit anti-rat haptoglobin and alpha2-macroglobulin IgG showing cross reactivity with albumin was simply eliminated.     

The identification of epitopic sites in human alpha 1-proteinase inhibitor.

Human alpha 1-proteinase inhibitor (alpha 1-PI) yielded nine fragments on cleavage with CNBr. The amino acid sequences of these fragments were determined. Three of these CNBr-cleavage fragments, namely fragment I (residues 64-220), fragment II (residues 243-351) and fragment III (residues 1-63), were found to bind rabbit polyclonal antibodies against chemically oxidized alpha 1-PI and mouse polyclonal antibodies against native alpha 1-PI by the Bio-Dot method (enzyme-linked immunosorbent assay on nitrocellulose). These fragments, I, II and III, inhibited by 60%, 25% and 5% respectively the binding between alpha 1-PI and the rabbit antibodies. Fragments I, II and III were subjected to proteolytic digestion, and 15, ten and five peptides were obtained from these fragments respectively. Only four of these peptides showed binding to the mouse antibodies against native alpha 1-PI. These were residues 40-63, 79-86, 176-206 and 299-323. A panel of monoclonal antibodies was prepared by conventional hybridoma technology, with chemically oxidized alpha 1-PI as the antigen. The ability of the monoclonal antibodies to bind native alpha 1-PI and CNBr-cleavage fragments I-III was determined. The monoclonal antibodies fell into three categories. Most (over 90%) belonged to group I, which was capable of binding alpha 1-PI and only fragment I. Antibodies in groups II and III bound alpha 1-PI and either fragment II or fragment III respectively. The ability of the peptides derived from proteolytic digestion of fragments I, II and III to bind three monoclonal antibodies representing each of the three groups was determined. Among all the peptides tested, only one (residues 176-206) derived from fragment I showed binding to the antibodies from group I, one (residues 299-323) derived from fragment II showed binding to the antibodies from group II, and one (residues 40-63) from fragment III showed binding to the antibodies from group III. Each of these three peptides also inhibited the binding between alpha 1-PI and the corresponding monoclonal antibodies. From these data we concluded that at least four epitopic regions (residues 40-63, 79-86, 176-206 and 299-323) were present in alpha 1-PI. Specific monoclonal antibodies to three of these sites were obtained.     

Fetuin and alpha-2HS glycoprotein induce alkaline phosphatase in epiphyseal growth plate chondrocytes.

A previously described chondrocyte alkaline phosphatase induction factor (CAP-IF) for chicken epiphyseal growth plate chondrocytes has been purified to SDS-PAGE homogeneity from fetal bovine serum by ammonium sulfate precipitation and by dye-ligand affinity (Affi-Gel Blue and Reactive Green-19 agarose) and hydroxyapatite column chromatographies. As determined by immunoprecipitation of [35S]methionine-labeled cellular proteins after 3 day treatment, this highly purified CAP-IF increases the level of AP and certain other membrane proteins 2- to 3-fold over control values. The pure protein of apparent 64.5 kDa molecular weight has been identified as fetuin by N-terminal amino acid sequencing. This was confirmed by the finding that high alkaline phosphatase (AP)-inducing activity is present in fetuin prepared by the Spiro method. However, fetuins prepared by the Pedersen or Deutsch procedures are inactive. At least half of the CAP-IF activity of fetuin was irreversibly destroyed by treatment with EDTA and addition of Zn2+ did not reactivate the EDTA-treated fetuin. Ascorbate synergistically enhanced the effect of fetuin on chondrocyte AP activity by over 8-fold during 3 day exposure. Because of the very high homology between fetuin and the A-chain of alpha 2-HS glycoprotein, we also tested and found that alpha 2HS glycoproteins from human serum and bovine bone are both strong AP inducers. Our findings suggest that the AP-inducing activity resides in a labile, cystatin/Zn(2+)-binding domain common to these related serum glycoproteins. These proteins appear to play a role in enhancing AP expression in normal growth plate cartilage differentiation.     

A fetuin-related glycoprotein (α2HS) in human embryonic and fetal development

Summary The human plasma protein, ₂HS glycoprotein, has an amino acid composition very similar to that of fetuin, the major protein in fetal calf and lamb serum. Immunohistochemical studies of human fetuses (6–33 weeks gestation) showed that ₂HS glycoprotein and fetuin have similar distributions in developing brain and several other tissues, e.g., bone, kidney, gonads, gastrointestinal tract, respiratory and cardiovascular systems. There were notable differences in the liver and thymus in the distribution of the two proteins. Fetuin and ₂HS glycoprotein are present in plasma and cerebrospinal fluid of both human and sheep fetuses; their concentrations are reciprocally related: in human plasma and cerebrospinal fluid ₂HS glycoprotein concentration is high and fetuin low; the reverse is the case in sheep fetuses.Estimates of the concentration of ₂HS glycoprotein in human fetal cerebrospinal fluid and plasma were obtained. It is suggested that ₂HS glycoprotein may play a role in developing tissues, especially in the human fetus, similar to that of fetuin in other species.     

The disulfide-rich region of platelet glycoprotein (GP) IIIa contains hydrophilic peptide sequences that bind anti-GPIIIa autoantibodies from patients with immune thrombocytopenic purpura (ITP)

Immune thrombocytopenic purpura (ITP) is an autoimmune blood disease caused by autoantibody-mediated destruction of blood platelets. Platelet glycoprotein (GP) IIb/IIIa is a common target for antiplatelet autoantibodies. The present studies were undertaken (1). to confirm whether the disulfide rich repeat region of GPIIIa contains target epitopes for antiplatelet antibodies in patients with ITP; (2). to determine whether these antigens were defined by peptide sequences in the absence of post-translational modification; and (3). to correlate observed immunologic reactivity with the recently solved X-ray crystallographic structure of an analogous integrin complex, the vitronectin receptor, alpha(V)beta(3). Recombinant fusion proteins of four GPIIIa extracellular sequences were prepared and purified. Immunoblotting results with purified recombinant peptides showed potent reactivity of 16 of 24 ITP patient serum anti-GPIIb/IIIa antibodies with the fusion protein containing the GPIIIa sequence of residues from 468 to 691. These results are consistent with a report by Kekomaki et al. that a 50 kDa chymotryptic digestion product of GPIIIa isolated from blood platelets contains target epitopes for serum antiplatelet antibodies in 16 of 33 ITP patients. Smaller peptides including residues 446-501 and residues 593-691 each reacted with only 5 of the 24 patient sera; furthermore all but 3 of these interactions were very weak. Visualization of the conformation of the extracellular portion of alpha(V)beta(3) reveals the location of the 222-residue antigenic GPIIIa (beta(3)) peptide 'B' at the immediately extracellular region of the protein that includes a beta-tail domain and several integrin-EGF domains. In summary, predictions of hydrophilicity, surface accessibility and antigenicity and the three dimensional structure of the beta(3) integrin correlate with autoantibody binding to a recombinant GPIIIa peptide 'B' containing residues 468-691.     

The complete amino acid sequence of rabbit muscle phosphoglucomutase

The complete amino acid sequence of rabbit muscle phosphoglucomutase has been determined by isolating the 11 peptide fragments produced by the cyanogen bromide cleavage reaction and subjecting these to automated sequencing procedures. Products produced by treatment of some of these fragments with hydroxylamine, iodosobenzoic acid, mild acid, cyanogen bromide in formic and heptafluorobutyric acids, Staphylococcus aureus V8 protease, and trypsin (with or without blocking at lysine residues) were used to complete the sequence for each of the cyanogen bromide fragments. The cyanogen bromide fragments were ordered by isolating the four tryptic peptides produced by a limited tryptic digest of the native enzyme in the presence of its substrates and its bivalent metal ion activator, Mg2+, degrading these by means of trypsin, after blocking digestion at lysine residues, and isolating and identifying all fragments thus produced that contained 10 or more residues. The 561-residue sequence thus obtained is one of the longest that has been determined by chemical means. There is excellent agreement between this sequence and published compositions after appropriate normalization. The absorbance of the enzyme is about 7.0 at 278 nm for a 1% solution; this value is 9% lower than that previously used.     

Proton magnetic resonance and binding studies of proteolytically modified neurophysins

The proton NMR spectra and role in peptide binding of carboxyl-terminal and NH2-terminal neurophysin residues were studied by preparation of bovine neurophysin-I derivatives from which residues 90-92 had been cleaved by carboxypeptidase or residues 1-8 excised by trypsin. The carboxypeptidase-treated protein showed normal peptide-binding behavior. NMR comparisons of this derivative and the native protein allowed identification of proton resonances associated with residues 89-92, confirmed a lack of functional role for this region of the protein, and permitted new observations on the behavior of neurophysin's aromatic residues. The trypsin-treated protein bound peptide with an affinity only 1/50 that of the native protein at pH 6 but evinced the same binding specificity and pH dependence of binding as the native protein. These results argued against direct interaction of residues in the 1-8 sequence with bound peptide and for a role for these residues, particularly Arg-8, in conformational stabilization of the active site; this role is held to be additional to the reported influence of 1-8 on dimerization. NMR comparisons of the trypsin product and native protein allowed preliminary assignment of a set of alkyl proton resonances to residues within the 1-8 sequence and were compatible with a restricted environment for Arg-8. Conformational differences between native and trypsin-treated proteins were manifest particularly by differences in the NMR spectra of Phe and Tyr-49 ring protons. The behavior of Phe ring protons was consistent with the reported decreased dimerization constant of the trypsin product and suggested participation of Phe-22 or -35 in dimerization. The behavior of Tyr-49 provided the first direct evidence of a change in secondary or tertiary structure associated with excision of residues 1-8. Suggested mechanisms by which this conformational change reduces binding include a direct effect on Tyr-49 and/or a conformational rearrangement of active site residues near Tyr-49.     

A predominant group-specific neutralizing epitope of human immunodeficiency virus type 1 maps to residues 342 to 511 of the envelope glycoprotein gp120.

Recombinant native human immunodeficiency virus type 1 (HIV-1) envelope glycoproteins gp160 and gp120 (residues 1 to 511) expressed in insect cells quantitatively adsorbed the group-specific neutralizing antibodies found in human sera. However, these antibodies were not adsorbed by envelope fragment 1 to 471 or 472 to 857 or by both fragments sequentially, even though together they add up to the full-length gp160 sequence. A hybrid envelope glycoprotein was constructed with residues 342 to 511 of the HIV-1 sequence and residues 1 to 399 of the simian immunodeficiency virus type 1 sequence to vary the HIV-1 sequence while preserving its conformation. This hybrid glycoprotein quantitatively adsorbed human neutralizing antibodies, while native simian immunodeficiency virus type 1 envelope glycoprotein did not. These results identify a new neutralizing epitope that depends on conformation and maps to residues 342 to 511 of gp120. It overlaps the extended CD4-binding site but is distinct from the V3 loop described previously (K. Javaherian et al., Proc. Natl. Acad. Sci. USA 86:6768-6772, 1989; J. R. Rusche et al., Proc. Natl. Acad. Sci. USA 85:3198-3202). Since it is conserved among diverse HIV-1 isolates, this new epitope may be a suitable target for future vaccine development.     

Ca2+ binding properties and Ca2(+)-dependent interactions of the isolated NH2-terminal alpha fragments of human complement proteases C1-r and C1-s

The NH2-terminal alpha fragments of human complement proteases C1-r and C1-s were obtained by limited proteolysis of the native proteins with trypsin, and isolated. C1-r alpha extended from residues 1 to 208 of C1-r A chain, with at least two cleavage sites within disulfide loops, after lysine 134 and arginine 202. C1-s alpha comprised residues 1-192 of the C1-s A chain, with one cleavage site within a disulfide loop, after arginine 186. C1-r alpha was monomeric either in the presence or absence of Ca2+ but formed Ca2(+)-dependent dimers with native C1-s. C1-s alpha dimerized in the presence of Ca2+ and formed Ca2(+)-dependent tetramers (C1-s alpha-C1-r-C1-r-C1-s alpha) with native C1-r. C1-r alpha and C1-s alpha associated in the presence of Ca2+ to form C1-r alpha-C1-s alpha heterodimers. Equilibrium dialysis studies indicated that each alpha region binds Ca2+ with a dissociation constant ranging from 19 microM (native proteins) to 38 microM (fragments). C1-r alpha, C1-r alpha-C1-s alpha, and the native C1-s-C1-r-C1-r-C1-s tetramer bound 0.9, 1.9, and 4.0 Ca2+ atoms/mol, respectively, whereas dimers C1-s alpha-C1-s alpha and C1-s-C1-s incorporated 2.9 and 3.0 Ca2+ atoms/mol. It is concluded that each alpha region contains one high affinity Ca2+ binding site. This 1:1 stoichiometry is maintained upon heterologous (C1-r-C1-s) interaction, whereas the homologous (C1-s-C1-s) interaction provides one additional binding site.     

Purification and characterization of Tora-bean (Phaseolus vulgaris) lectin

A lectin purified from the Tora-bean (Phaseolus vulgaris) by affinity chromatography with Con-A Sepharose was shown to be a glycoprotein containing 7.8% neutral sugars (D-mannose, N-acetyl-D-glucosamine, L-fucose, and D-xylose, in a molar ratio of 9.6 : 2.0 : 0.6 : 0.7). Its molecular weight was 130,000, as estimated by exclusion gel chromatography, and SDS gel electrophoresis showed that it consists of four subunits of molecular weight 32,000. The lectin reacts with various glycoproteins, i.e., blood group substances, human parotid salivary glycoprotein, fetuin, and bovine submaxillary mucin. Divalent cations, such as Ca2+, Mn2+, and Mg2+, appear to stimulate its reactivity. Inhibition tests using the glycopeptide fragment from fetuin and some oligosaccharides, as well as the binding test with 14C-N-acetyl-lactosamine suggest that the sequence of D-galactose, N-acetyl-D-glucosamine, and D-mannose residues in the carbohydrate chain of fetuin is essential for binding.