ASPARAGINE-LINKED OLIGOSACCHARIDES ARE LOCALIZED TO SINGLE EXTRACYTOSOLIC SEGMENTS IN MULTISPAN MEMBRANE-GLYCOPROTEINS

A comprehensive survey of mammalian multi-span (polytopic) membrane pr oteins showed that asparagine(N)-linked oligosaccharides are localized to single extracytosolic segments. In most membrane proteins this is because potential consensus sites for N-glycosylation (Asn-Xaa-Ser/Thr , X not equal Pro) are not found in multiple extracytosolic segments. In functional proteins where consensus N-glycosylation sites are conta ined within more than one extracytosolic segment, only the first segme nt contains N-linked carbohydrate. An exception is the alpha-subunit o f the Na+ channel, which consists of a duplicated structure containing two glycosylated segments. The average size of established N-glycosyl ated loops connecting two transmembrane segments is 62 residues, with the smallest glycosylated loop being 33 residues in size. N-glycosylat ed sites are more highly conserved than nonglycosylated (primarily cyt osolic) sites and are more common toward the N-terminus of the membran e domain of multi-span membrane proteins. The optimal conditions for g lycosylation of consensus sites within an extracytosolic domain of a m ulti-span membrane protein are (i) the acceptor site is well-spaced (g reater than 10 residues) from the transmembrane domain, (ii) the loop is greater than 30 residues in size and (iii) the segment is the first in the protein to contain a suitable extracytosolic consensus site. T he localization of N-linked oligasaccharide chains to a single protein segment suggests either glycosylation of multiple loops may compromis e protein folding or function, or only a single polypeptide domain can be optimally glycosylated during biosynthesis in vivo.