Preferential transfer of the complete glycan is determined by the oligosaccharyltransferase complex and not by the catalytic subunit

Abstract

Most eukaryotic cells show a strong preference for the transfer in vivo and in vitro of the largest dolichol-P-P-linked glycan (Glc 3Man9GlcNAc2) to protein chains over that of biosynthetic intermediates that lack the full complement of glucose units. The oligosaccharyltransferase (OST) is a multimeric complex containing eight different proteins, one of which (Stt3p) is the catalytic subunit. Trypanosomatid protozoa lack an OST complex and express only this last protein. Contrary to the OST complex from most eukaryotic cells, the Stt3p subunit of these parasites transfers in cell-free assays glycans with Man 7-9GlcNAc2 and Glc 1-3Man9GlcNAc2 compositions at the same rate. We have replaced Saccharomyces cerevisiae Stt3p by the Trypanosoma cruzi homologue and found that the complex that is formed preferentially transfers the complete glycan both in vivo and in vitro. Thus, preference for Glc 3Man9GlcNAc2 is a feature that is determined by the complex and not by the catalytic subunit. N-glycosylation ͉ Saccharomyces cerevisiae ͉ Trypanosoma cruzi

The oligosaccharyltransferase subunits OST48, DAD1 and KCP2 function as ubiquitous and selective modulators of mammalian N-glycosylation

“…Indeed, higher eukaryotes seem to have acquired additional non-catalytic subunits, as exemplified by KCP2, that are over and above known and predicted homologues of the well-defined yeast OST subunits (Kelleher and Gilmore, 2006;Shibatani et al, 2005). Conversely, various kinetoplastid single-component OSTs display the capacity to N-glycosylate an array of substrates (Castro et al, 2006;Izquierdo et al, 2009;Kelleher and Gilmore, 2006;Nasab et al, 2008). Thus, the idea that OST complexity directly reflects substrate complexity may be over simplistic, and non-catalytic OST subunits may perform other roles that remain to be delineated.…”

“…Whilst eukaryotes such as trypanosomatids and giardia are able to N-glycosylate newly synthesised polypeptides using a singlecomponent enzyme, a member of the STT3 family (Castro et al, 2006;Izquierdo et al, 2009;Kelleher and Gilmore, 2006), many eukaryotic OSTs consist of multiple subunits. The composition and subunit organisation of the mammalian enzyme is particularly complicated, having two homologues of Saccharomyces cerevisiae Stt3p, STT3A and STT3B.…”

Architecture and Biosynthesis of the Saccharomyces cerevisiae Cell Wall

“…Evidence for a role of complex subunits other than Stt3 was obtained with Trypanosoma cruzi Stt3, which transfers GlcNAc 2 Man 7--9 to protein in vitro as efficiently as it does glucosylated oligosaccharides. When expressed in S. cerevisiae in place of native Stt3, trypanosomal Stt3 now preferentially transferred GlcNAc 2 Man 9 Glc 3 to protein in vitro and in vivo (Castro et al 2006). Similarly, when Leishmania Stt3 is expressed in the context of the other S. cerevisiae OST subunits, the Leishmania protein acquires a preference for transferring glucosylated oligosaccharides, rather than the non--glucosylated oligosaccharides that it transfers in the protist itself (Hese et al 2009).…”

Polypeptide binding specificities of Saccharomyces cerevisiae oligosaccharyltransferase accessory proteins Ost3p and Ost6p

“…4,12 The various enzymes from a single organism can have different glycan and protein substrate specificities, such that gene duplication and divergence may have increased the catalytic range of efficient glycosylation. [13][14][15][16][17] Higher eukaryotes have evolved multiprotein complex OTases, in which subunits are involved in selection of mature glycan substrate and regulation of activity. [18][19][20] Duplication and divergence of multiprotein OTase subunits has also occurred, with genes encoding Stt3p and Ost3/6p proteins present in two copies in some organisms.…”