Previously, we have characterized the HIV-I SF2 gp120 glycopeptides using matrix-assisted laser desorption/ionization mass spectrometry (MALDI/MS) and nanospray electrospray ionization (ESI). Although we characterized 25 of 26 consensus glycosylation sites, we could not obtain any information about the extent of sialylation of the complex glycans. Sialylation is known to alter the biological activity of some glycoproteins, e.g., infectivity of some human and nonhuman primate lentiviruses is reduced when the envelope glycoproteins are extensively sialylated, and thus, characterization of the extent of sialylation of complex glycoproteins is of biological interest. Since neither MALDI/MS nor nanospray ESI provided much information about sialylation, probably because of suppression effects inherent in these techniques, we utilized online nanocapillary high performance liquid chromatography (nHPLC) with ESI/MS to characterize the sites and extent of sialylation on gp120. Eight of the known 26 consensus glycosylation sites of HIV-I SF2 gp120 were determined to be sialylated. Two of these sites were previously uncharacterized complex glycans. Thirteen high mannose sites were also determined. The heterogeneity of four of these sites had not been previously characterized. In addition, a peptide containing two consensus glycosylation sites, which had previously been determined to contain complex glycans, was also determined to be high mannose as well. (J Am Soc Mass Spectrom 2004, 15, 1545-1555

Monoclonal antibody (MAb) 6-11A directed against Streptococcus mutans surface adhesin P1 was shown previously to influence the mucosal immunogenicity of this organism in BALB/c mice. The specificity of anti-P1 serum immunoglobulin G (IgG) and secretory IgA antibodies and the subclass distribution of anti-P1 serum IgG antibodies were altered, and the ability of elicited serum antibodies to inhibit S. mutans adherence in vitro was in certain cases increased. MAb 6-11A is known to recognize an epitope dependent on the presence of the proline-rich region of the protein, although it does not bind directly to the isolated P-region domain. In this report, we show that MAb 6-11A recognizes a complex discontinuous epitope that requires the simultaneous presence of the alanine-rich repeat domain (A-region) and the P-region. Formation of the core epitope requires the interaction of these segments of P1. Residues amino terminal to the A-region also contributed to recognition by MAb 6-11A but were not essential for binding. Characterization of the MAb 6-11A epitope will enable insight into potential mechanisms of immunomodulation and broaden our understanding of the tertiary structure of P1.