Comp. Biochem. Physiol. Vol. 116B, No. 4, pp. 423–430, 1997 ISSN 0305-0491/ 97/$17.00 Copyright  1997 Elsevier Science Inc. PII S0305-0491(96)00264-7 The Carbohydrate Moiety of IgM From Atlantic Salmon (Salmo salar L.) B. Magnado ´ttir, B. K. Gudmundsdo ´ttir and S. Gudmundsdo ´ttir Institute for Experimental Pathology, University of Iceland, Keldur v/Vesturlandsveg, IS-112 Reykjavı ´k, Iceland ABSTRACT. The carbohydrate moiety of salmon IgM was estimated to be about 12.5% of the total molecular weight of salmon IgM based on SDS-PAGE analysis and 8.0% based on FACE analysis. The carbohydrate moiety was restricted to the heavy chain and was all N-linked. Six different oligosaccharides were identified using the FACE oligosaccharide profiling technique. Monosaccharide composition analysis, as well as digestion with endoglycosidase H, suggested that the oligosaccharides were mainly of the complex type rather than high mannose type. Removal of about 80% of the carbohydrate affected the sensitivity of IgM to trypsin but had no effects on antigen binding or the complement fixation ability of anti s-RBC IgM. comp biochem physiol 116B; 4:423–430, 1997.  1997 Elsevier Science Inc. KEY WORDS. Endo H glycosidase, FACE, glycosylation, IgM, PNGase, F, O-Glycanase, Salmo salar, trypsin INTRODUCTION munoglobulins: they may contribute to their structural sta- bility and protect against proteases (33), they may be in- Glycosylation represents a major post-translational modifi- volved with effector functions like complement fixation and cation of proteins that can influence their structure and Fc receptor recognition (16,22,27,29,36), glycosylation can function. All immunoglobulins are glycosylated, and the influence the avidity (5) and polymerization of IgM (11,26) amount of carbohydrate present and the nature of glycosyla- and changes in the oligosaccharide moiety are sometimes tion are characteristic of each immunoglobulin class (13). associated with ageing, pregnancy or diseases (4,8). Most of The two main types of oligosaccharide linkages to the these conclusions have been reached from studies of immu- protein are the N-linked and O-linked types (15). N-linked noglobulins from mammalian species that possess several Ig oligosaccharide is covalently bonded through nitrogen to classes with different structure and function, including IgG, asparagine (Asn) when it occurs in the sequence Asn-X- IgA and pentameric IgM. Ser/Thr, where X may be any amino acid other than pro- The immune system of fish is in many ways similar to the line. O-linked oligosaccharide is covalently bonded through mammalian system. There are, however, certain differences oxygen to serine and threonine but the amino acid sequence due to their position on the evolutionary ladder, their poiki- where bonding occurs is not defined. The possible structural lothermic nature and adaptation to the aquatic environ- diversity of the oligosaccharide is enormous. There are, for ment (14). Most fish species have only one immunoglobulin example, several subgroups of N-linked oligosaccharides class, tetrameric IgM. Exceptions are the chondrichthyan that share a common core structure but vary with respect species, which possess pentameric IgM and a low-molecular- to the composition and complexity of their outer chains weight immunoglobulin of separate genetic origin (17,21). (12,15,23,30). Very few reports exist in the literature about the glycosyla- Concurrent with the development of protein engi- tion of fish IgM (1,10). neering, the importance of the normal glycosylation of the Information about the likely N-linked glycosylation of recombinant protein has become clearly apparent (35). It IgM from fish and higher vertebrates can be gained by exam- has also become apparent that the effects of glycosylation ining the amino acid sequence analysis of IgM subunits, cannot be generalised but must be examined on a case-by- where this is available (2,3,7,17,19,37). Such an examina- case basis (12,30,35). However, certain roles are commonly tion shows that normally only the heavy chain is glycosyl- attributed to the oligosaccharide moiety of, for example, im- ated, commonly bearing five theoretical sites that are re- stricted to the constant regions. However, there are two Address reprint requests to: B. Magnado ´ttir, Institute for Experimental Pa- noticeable differences in their distribution on IgM heavy thology, University of Iceland, Keldur v/ Vesturlandsveg, IS-112 Reyk- chain of fish and higher vertebrates. First, fish μ chain does javı ´k, Iceland. Tel. 354-567-4700; Fax 354-567-3979; E-mail: bergmagn@ not seem to have a glycosylation site on the Cμ1 domain rhi.hi.is. Received 24 June 1996; accepted 2 October 1996. in the Fab region. This applies to both tetrameric IgM from