Synthesis and biological activity of the extracellular Ig domain of emmprin carrying various carbohydrate chains

摘要

Emmprin is a glycoprotein located on the surface of tumor cells.It stimulates nearby fibroblasts to produce matrix metalloproteinases(MMPs),which play essential roles for tumor invasion and metastasis.Emmprin is heavily glycosylated and is not functional without the carbohydrate moiety.This protein consists of two extracellular immunoglobulin(Ig)-like domains,transmembrane and short intracellular domain.The functional site is the extracellular first Ig domain,which has an N-glycosylation site at Asn~(44).However,the effect of the N-glycosylation on its activity is not fully understood.We have already prepared first Ig domain(34-94)carrying GlcNAc_n(n=0-2)at Asn~(44)by the thioester method.The biological study shows that MMP stimulation activity increases as the sugar chain becomes longer from n=l to 2,whereas Ig domain itself(n=0)has no activity,demonstrating the importance of the carbohydrate moiety.We also prepared the Ig domain carrying N-linked core pentasaccharide(Man_3GlcNAc_2),the activity of which is under investigation.However,CD analysis indicates that all these domains assume no particular secondary structure,though the distinct difference in spectrum exists between the glycosylated and the non-glycosylated domains.To clarify the function of this glycodomain from the structural point,more stable conformation has to be formed.Thus,we intended to increase the conformational stability by extending the peptide chain to the C-terminus.In this study,a peptide composed of two extracellular Ig domains(34-192)carrying the(GlcNAc)_2 at Asn~(44)was synthesized and its conformation was characterized by CD measurement.