MINOR INFLUENCE OF SIALIC-ACID ON CONFORMATION OF A MEMBRANE-BOUND OLIGOSACCHARIDE RECOGNITION SITE

Wideline H-2 NMR spectroscopy was used to assess the conformational an d orientational effects of N-acetylneuraminic acid (NeuAc) (sialic aci d) as a component of a particular oligosaccharide chain at a bilayer m embrane surface. For this purpose, three glycosphingolipids, sharing a neutral core tetrasaccharide and differing only in the number of sial ic acid residues, were compared. The starting compound was GD(1A), whi ch has terminal sialic acid attached to the second and fourth sugars o f its neutral tetrasaccharide core. GD(1A) was probe-labeled in a nonp erturbing fashion on both of these sialic acid residues and on its sin gle GalNAc residue by replacement of -COCH3 with -COCD3, giving [(d(3) NeuAc)(2),d(3)-GalNAc]GD(1A). This represents the most complex glycoli pid to have been studied by H-2 NMR spectroscopy at a bilayer membrane surface. The sialic acid residue on the fourth sugar from the membran e was subsequently removed to produce the glycolipid [d(3)NeuAc,d(3)Ga lNAc]GM(1) deuterated at the two remaining amino sugars. The neutral g lycolipid [d(3)GalNAc]asialo-GM(1) was then generated by removal of th e second sialic acid residue, leaving an uncharged species deuterated at one (internal) oligosaccharide chain site (GalAc). The effect of si alic acid was further examined by selective deuteration of GM(1) and a sialo-GM(1) at C-6 of the terminal Gal residue, giving [d(2)Gal]GM(1) and [d(2)Gal]asialo-GM(1). Spectra of the three glycosphingolipids wer e compared at 7.7 mol % in unsonicated fluid bilayers of 1-palmitoyl-2 -oleoylphosphatidylcholine containing 23 mol % cholesterol. For liposo mes suspended in buffered salt solutions with 2 mM Ca2+, H-2 NMR spect ra demonstrated the presence of a well defined average conformation fo r each oligosaccharide chain. This preferred average conformation pers isted over a wide temperature range, consistent with there being a sin gle major oligosaccharide conformer in each case. Spectral features ar ising from both deuterated amino sugars of GM(1) were present, essenti ally unchanged, in spectra of GD(1A) Features associated with the sing le deuterated amino sugar (GalNAc) of asialo-GM(1) could be identified , little changed, in spectra of GM(1) and GD(1A). Similarly, deuterons in the terminal Gal residue of asialo-GM(1) produced the same spectru m seen for this residue in GM(1). Our findings indicate that certain m ajor conformational and orientational features of this complex oligosa ccharide recognition site are preserved, within a maximum angular devi ation of +/-5 degrees or less, upon addition or removal of a sialic ac id residue.