Structural studies of the glycopeptides of B-chain of cinnamomin--a type II ribosome-inactivating protein by nuclear magnetic resonance.

Abstract

Cinnamomin is a plant type II ribosome-inactivating protein (RIP) isolated from the seeds of Cinnamomum camphora. It consists of two nonidentical polypeptide chains (A- and B-chain) held together through one disulfide linkage. Its A- and B-chain contain 0.3% and 3.9% sugars respectively. The B-chain of cinnamomin was digested by pronase E and then the liberated glycopeptides were separated from non-glycopeptides by gel filtration chromatography on a Bio-Gel P-4 column. Three crude glycopeptides were obtained by continuing chromatography over anion-exchange resin (AG1-X2) in the buffer of 2% pyridine-acetic acid (pH 8.3) with a polygradient elution system. Through further purification by the gel filtration chromatography and HPLC, three major glycopeptides, GP1, GP2 and GP3 were obtained. Mainly by two-dimensional Nuclear Magnetic Resonance (NMR) including TOCSY, DQF-COSY, NOESY, HMQC and HMBC, their primary structures were analyzed as: Man\balpha1,3Man\balpha1,6(Man\balpha1,3)(Xyl\bbeta1,2)Man\bbeta1,4GlcNAc\bbeta1,4GlcNAc\bbeta1-(Gly-)Asn-Asn-Thr(GP1), Man\balpha1,6(Man\balpha1,3)(Xyl\bbeta1,2)Man\bbeta1,4GlcNAc\bbeta1,4(Fuc\balpha1,3)GlcNAc\bbeta1-Asn-Ala-Thr(GP2),Man\balpha1,6(Man\balpha1,3)Man\balpha1,6(Man\balpha 1,2 Man\balpha1,3)Man\bbeta1,4GlcNAc\bbeta1,4GlcNAc\bbeta1-(Ala-)Asn-Gly-Thr(GP3).