Cell-associated episialin is a complex containing two proteins derived from a common precursor.

M.J Ligtenberg,L Kruijshaar,F Buijs,M Van Meijer,S.V Litvinov,J Hilkens

doi:10.1016/s0021-9258(18)42677-4

Abstract

cDNA for the epithelial sialomucin episialin encodes a transmembrane molecule with a large extracellular domain, which mainly consists of repeats of 20 amino acids. Here we confirm the existence of a previously proposed proteolytic cleavage of episialin that occurs in the endoplasmic reticulum (Hilkens, J., and Buijs, F. (1988) J. Biol. Chem. 263, 4215-4222) and show that a similar cleavage takes place in in vitro translation systems. Using in vitro translation of truncated mRNAs, we map the cleavage site to a region located between 71 and 53 amino acids upstream of the transmembrane domain. Analysis of a mutant, in which this region has been deleted, indicates that the cleavage sites used in vitro and in vivo are identical or in close proximity. Both cleavage products remain associated although they are not linked through disulfide bonds. Therefore, the subunit derived from the N terminus, which represents the actual mucin-like domain, remains indirectly anchored to the cell membrane as a result of its interaction with the C-terminal subunit.

Highlights

We confirm the existence of a previously acids at the protein level, variant A being nine amino acids proposed proteolyticcleavage of episialin that occurs longer than variant B. in the endoplasmic reticulum (Hilkens, J., and Buijs, The biosynthesis of episialin has been described by several bFtmsmtrhiieeo.tRegaten(mwitsN1ose9bunaAye8rshssnesat8a,dien7mw)smeJi1benise.lada.mevBnornUidaimtdoprcs6leiaol.netl3iehaanCgtanev.ehmidadcneAg,lmiieintennvnaa.oaidkvt2lvriyeaa6ciossgcva3oiipoestt,fderals4sasaari2tctneuhem1esipa6ldtuatiso-netttt4hnriaia2oneetnn2airtcm2,evaog)iflitinooroatofrncrnwtulrdtelinhhaonasciencvhcaatcshaothtlltegrwaoeidase-sdne s-gdHrKt(ae1reuikso9tluefeku8eclep8t,tnps)ao1hlsb(fa9aHlaca8eveni9epldp)kori.roebBnBetnscoeuestuotirhhjrvlsayseenHot(did1reicBl9nkci8udsetl8eihonja)rspasevh,ltdaaaat1ungsvh9mcedee8e.ipB8dTcr;muhobrLiipoejyssilotneips(c2csu1eulu0det9llayu8attekhr8mitDv)aam,eaatln.asct,dwislh1nesi9iLacst8veohis8nfaihn;gsititleAfhe4tysbeoheicemsofcatiuunirtanlhsrdldst..e proximity
The cDNA sequence predicts that episialin is a type I actual mucin-like domain containing the repetitive region membrane molecule with a large extracellular domain, which from the transmembrane and the cytoplasmic domain
The major part of the extracellular domain released fromcarcinoma cells and found in serum of patients consists of repeats of 20 amino acids that have a high content with breast cancer, in which it is demonstrated to be an of serine and threonineresidues, many of which are potential important marker to monitor breast cancer therapy

Summary

MATERIALS AND METHODS

CDNA Constructs-The generation of full-length cDNAs encoding episialin has been described previously (Ligtenberg et al, 1992). Of the Netherlands Red Cross Blood Transfusion Service, Plesman- cDNA constructscontaining a single repeat were generated from laan 125,1066 CX Amsterdam, The Netherlands. W Supported by a fellowship from the International Union for ing a BsmI-EcoNI fragment of the cDNA constructs with a BsmI-. The abbreviations used are: SDS, sodium dodecyl sulfate; LRP, 205122018; Fax: 31-206172625.

Formation Complex Episialin

RESULTS

WM kDa

Evidencefor the Association of the Cleavage Productsin

DISCUSSION