Pergamon 0161-5890(95)00132-8 zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA Molecular Immunology. Vol. 33. No. 1, pp. 15-24, 1996 Copyright ;i;! 1996 Elsevier Science Ltd. All rights reserved Printed in Great Britain 0161-5890196 $15.00 + 0.00 IgM POLYMERIZATION INHIBITS THE GOLGI-MEDIATED PROCESSING OF THE p-CHAIN CARBOXY-TERMINAL GLYCANS MARIE-MADELEINE CAL&* SILVIA GUENZI.* STEPHANA CARELLI,* THOMAS SIMMEN,* ANTONELLA SPARVOLI* and ROBERTO SITIAP; *DIBIT San Raffaele Scientific Institute, 20132 Milano, Italy: tIST, 16132 Genova. Italy (First receiced 14 June 1995; accepted in revisedform 12 September 1995) Abstract-Secreted glycoproteins generally contain oligosaccharides of the complex type. However, several molecules have been described in which individual glycans are processed differently from one another. Folding, assembly and oligomerization could affect the maturation of certain glycans by hindering them to the Golgi processing machinery. We have tested this possibility by analysing a panel of engineered murine p chains secreted as ~2L2 monomers or as polymers, and having or not the carboxy-terminal glycan (Asn563). In secreted IgM polymers, Asn563 bears high-mannose oligosaccharides, typical of endoplasmic reticulum resident proteins, while complex sugars are found at the other four sites (Brenckle and Kornfeld. 1980 Arch. Biochem. Biophys. 243,605-618). Polymeric and monomeric IgM contain p chains whose glycans are processed differently. We show here that this is mainly due to the differential processing at the Asn563 glycan, which undergoes Golgi- mediated processing when IgM are secreted in the monomeric form. These results indicate that the oligomerization-dependent accessibility to the sugar modifying enzymes can be one of the key features that dictate the extent of oligosaccharide processing in multimeric glycoproteins. The presence of high mannose glycans at Asn563 implies that IgM polymerization takes place before encountering mannosidase II. likely in a pre-Golgi compartment. Kq, words: glycosylation. IgM. intracellular transport. processing, oligomerization, reducing agents, secretion. INTRODUCTION In general, secreted proteins contain N-linked glycans of the complex type [see Kornfeld and Kornfeld (1985) for a review]. These are generated from the Glu,Man,GlcNAc, precursors by a series of glycosidases and glycosyl-trans- ferases spatially distributed so as to allow the sequential processing while glycoproteins are transported along the exocytic pathway (Roth and Berger, 1982; Kornfeld and Kornfeld, 1985; Nilsson et al., 1993). However, in some secreted glycoproteins, certain glycans are not processed and remain in the high-mannose state [Man,,GlcNAcz] characteristic of ER resident proteins (see Kornfeld and Kornfeld, 1985: Faye et al.. 1986 and references therein). One such example is IgM, a polymeric immunoglobulin secreted by plasma cells. In both lymphoid cells and non- lymphoid transfectants, secretion of IgM is restricted to covalent polymers ([~2L2], + J or [~2L2&) (Cattaneo and Neuberger, 1987; Davis et a/., 1989a; Randall et al.. $Author to whom correspondence should be addressed at: Molecular Immunology, DIBIT-HSR, Via Olgettina 58. 20 132 Milano. Italy. Abbrer:iations: ER, endoplasmic reticulum, Gal, galactose. GlcNAc, N-acetylglucosamine. Ig, immunoglobulin, Man, mannose. 2ME. 2-mercaptoethanol. 1992). Unpolymerized intermediates are retained intra- cellularly by disulfide interchange reactions involving the carboxy-terminal cysteine (Cys575) of p chains (Sitia et al., 1990; Alberini el al., 1990). The Cys575 also mediates the covalent assembly (Davis et al.. 1989b; Sitia et al., 1990) and the intracellular degradation (Fra er al., 1993; Fra and Sitia, 1993) of unpolymerized IgM subunits. Although some contradictory reports have appeared in the literature (Shachar et al., 1992). IgM polymerization is thought to take place in the ER or in a related com- partment (Tartakoff and Vassalli, 1979; Sitia et al., 1987, 1990; Fra et al., 1993; Brewer et al., 1994; Bornemann et al., 1995). The five glycans present on secreted murine p chains have been characterized by both biochemical and NMR analyses. While asparagines 171, 332 and 364 bear com- plex glycans (Brenckle and Kornfeld, 1980; Anderson et al., 1985). conflicting reports have appeared concerning the state of Asn402: Sun et al. (1991) described the pres- ence of hybrid oligosaccharides, the most frequent form in human p chains (Chapman and Kornfeld, 1979a Chapman and Kornfeld, 1979b), while others found a complex oligosaccharide (Brenckle and Kornfeld, 1980; Anderson et al., 1985). In contrast, the vast majority of IgM polymers secreted by cultured cells bear a high mannose (man, 8) glycan at Asn563 (Brenckle and 15