Abstract
We have previously reported that concanavalin A (ConA) is precipitated by a high mannose type glycopeptide (Brewer, C. F. (1979) Biochem. Biophys. Res. Commun. 90, 117-122; Bhattacharyya, L., and Brewer, C. F. (1986) Biochem. Biophys. Res. Commun. 137, 670-674). In the present study, we have investigated the ability of a series of high mannose and bisected hybrid type glycopeptides to bind and precipitate the lectin. The modes of binding of the glycopeptides were studied by nuclear magnetic relaxation dispersion (NMRD) techniques, and their affinities were determined by hemagglutination inhibition measurements. The stoichiometries of the precipitation reactions were investigated by quantitative precipitation analysis. The equivalence zones (regions of maximum precipitation) of the precipitin curves indicate that certain high mannose and bisected hybrid type glycopeptides are bivalent for lectin binding. From the NMRD and precipitation data, we have identified two protein binding sites on each glycopeptide: one site on the alpha(1-6) arm of the core beta-mannose residue involving a trimannosyl moiety which binds with high affinity (primary site); and the other site on the alpha(1-3) arm of the core beta-mannose residue involving an alpha-mannose residue(s), which binds with lower affinity (secondary site). These two types of sites bind to ConA by different mechanisms. Certain bisected hybrid type glycopeptides were found to possess only the primary ConA binding sites, but not the secondary sites, and hence were able to bind but not precipitate the lectin. Other related glycopeptides have only the secondary type sites and thus exhibit low affinity and are unable to precipitate the protein. The results are related to the possible structure-function properties of cell-surface glycopeptides.
Highlights
From the $Departments of Molecular Pharmacology, and Microbiology and Immunology, Albert Einstein College of Medicine, Bronx, New York 10461 and SCentro Ricerche Sclavo, Via Fwrentina 1,53100 Siena, Italy
We have previously reported that concanavalin A of biological functions, including cell-cell recognition, and (ConA) is precipitated by a high mannose type glyco- migration of cells to particular organs, including the metaspeptide
The results indicate that certain high mannose and bisected hybrid type glycopeptides are bivalent and capable of precipitating the lectin
Summary
The principal finding in this study is that thehigh mannose type glycopeptides AC-CB, D3, andE3 (Fig. 1) andthe bisected hybrid type glycopeptide C3b (Fig. 2) are capable of binding and precipitating ConA. The results of the present study show that the disaccharides, 3-O-(a-D-mannOpyranOSyl)-D-mannOSaned 6 - 0 - ( a - ~ mannopyranosy1)-D-mannosew,hich are part of the structure of I , do not possess its high affinity binding (Table I) Both disaccharides induce changes in the NMRD profile of CMPL(not shown) similar toa-MDM. These results strongly argue for extended site binding of the two nonreducing mannose residues of 1, in agreement with our previous conclusions (8,9). AC-BA and AC-A fail to precipitate the protein under the same conditions as C3b and thehigh mannose type glycopeptides These results indicate that AC-BA and AC-A have the primary binding sites, but not the second sites. The @1( -4) substituted mannose residues in the a(1-3) arms of AC-BA and AC-A are no longer able to TABLEI
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