Supplementary Materialscb7b00797_si_001. examined. Endocytic receptors, including the mannose receptor, DC-SIGN, langerin, and DC-SIGNR (L-SIGN), interact predominantly with mannose-containing Rabbit Polyclonal to EPHA3 caps found on the mycobacterial polysaccharide lipoarabinomannan. Some of these receptors also interact with phosphatidyl-with Langerhans cells is mediated in part by binding of langerin to mannose-containing O-linked glycans on superoxide dismutase.47 The relatively strong signals observed for simple, terminal mannose residues are consistent with binding of langerin to the small glycans associated with mycobacterial glycoproteins.48 Dectin-2, which SRT1720 tyrosianse inhibitor binds to mycobacterial LAM,49 interacts with the Man1C2Man disaccharide.50 Structural analysis, combined with the ability of dectin-2 to bind yeast mannans and selected bacterial polysaccharides, indicates that this disaccharide motif can be either at a nonreducing terminus SRT1720 tyrosianse inhibitor or internally in a polysaccharide. The binding site can accommodate terminal mannose residues in other linkages, but at reduced affinity. These features are consistent with enhanced binding of cap structures on LAM that contain Man1C2Man (3, 4, and 6) with lower levels of binding to other mannose-containing compounds (Figure ?Figure66B). The absence of binding to 7 and 9 is consistent with structural data showing that derivatization of the 4-OH group of the nonreducing end mannose in Man1C2Man results in a steric clash.50 The sinusoidal endothelial cell receptor DC-SIGNR binds to a specific subset of mannose-containing glycans (Figure ?Figure66C). All of the strongest signals are for glycans with 1C2-linked mannose units, consistent with evidence that Man1C2Man is the preferred disaccharide ligand51 and that DC-SIGNR shows restricted binding to mammalian oligosaccharides compared to DC-SIGN.39 The difference in specificity likely derives from subtle differences in the binding site that restrict access by many oligosaccharides in DC-SIGNR. Mincle Binding to a definite Group of Mycobacterial Glycans The fluorescently tagged mincleCstreptavidin complex could be recognized directly or following a addition of a second antibody, with identical results (Shape ?Shape77A). The indicators for multiple glycans for the array that carry a number of non-reducing terminal mannose or glucose residues have become little in comparison to those for ligands including trehalose. Therefore, the binding specificity can’t be basically described based on an individual terminal monosaccharide residue but depends upon the current presence of the trehalose disaccharide.52 The need for binding of mincle to trehalose dimycolate (cord factor) is well-documented, but testing against the entire array provides several novel insights that are summarized in Shape ?Figure77B. The trehalose-containing glycans 38, 39, 54, and 55 bind regardless of the variation in substituents strongly. Open in another window Shape 7 Binding of mincle to mycobacterial glycans. (A) Mincle complexed with Alexa Fluor 488-conjugated streptavidin was utilized to probe the array at 5 g mLC1 and was recognized directly by dimension of fluorescence (remaining) or after further incubation having a Cy3-tagged anti-streptavidin antibody (ideal). (B) Schematic diagram from the binding sites in mincle as well as the positions occupied by person monosaccharide residues in oligosaccharide ligands. X represents either additional monosaccharide BSA or residues to that your oligosaccharide is conjugated. Residues in green shaded sites make beneficial interactions with the top of mincle; residues in yellowish regions SRT1720 tyrosianse inhibitor project from the top, and residues in reddish colored areas would clash with the top. (C) Model for binding of ligands including trehalose extended for the 6-OH group. (D) Style of Glc1C4Glc di- and trisaccharides bound to mincle. (E) Style of Glc1C6Glc disaccharides bound to mincle. The crystal structure of trehalose monobutyrate certain to bovine mincle (Proteins Data Standard bank entry 4ZRV) was utilized to magic size trehalose derivatives certain to mincle using PyMOL. Conformations of glycans, extracted from little molecule databases, weren’t modified, but unimportant regions were eliminated. Superpositions of specific monosaccharide residues, referred to at length in Supporting Info 1, had been performed manually. In sections E and D, parts of positive potential on the top of mincle are coloured blue, parts of adverse potential are coloured red, as well as the destined Ca2+ can be colored magenta. In the ligands, carbon atoms are colored green or orange and oxygen atoms are colored red. Glycans 54 and 55 represent surface lipooligosaccharides found in em Mycobacterium kansasii /em , an opportunisitic pathogen, but not in em M. tuberculosis /em .22 Binding of these glycans by mincle suggests that the binding site can accommodate additions to the 4-OH of one of the glucose residues in trehalose. The 4-OH of the glucose.