N-Glycosylation - The Sugar Code - page 89

chains are N - glycosylated by oligosaccharyltransferase ( OST ), a multisubunit
enzyme complex, at the -amino group of six asparagine residues in the consensus

sequences Asn- Xaa (≠ Pro) - Thr/Ser - Xaa (≠ Pro) (Asn sequon). Asn sequons are highly
underrepresented in glycoproteins, although they are often well preserved during
evolution. This refl ects their local infl uences during protein folding and assembly
[2]. Particular oligosaccharide structures modulate functional properties of mem-
brane proteins, such as those forming membrane channels, and adhesion mole-
cules, including NCAM1 (please see Chapter 30.6 - 8 for further details). In addition,
the N -glycans physically stabilize the conformation of mature proteins.

β

6.2
Initial Steps in Asparagine- Linked Glycosylation

N - Linked glycan ( N -glycan) biosynthesis starts with the transfer of preassembled
lipid - linked oligosaccharide ( LLO ) to the polypeptide (Figure 6.2). The LLO is
synthesized on dolicholpyrophosphate ( Dol - PP ) by the asparagine - linked g lycosyl-
ation (ALG) gene products at both sides of the ER membrane. Each of the con-
secutively acting glycosyltransferases uses as acceptor a specifi c LLO intermediate
and the respective donor substrate to guarantee accurate assembly of the LLOs.
All steps of this biosynthetic pathway up to Dol-PP-GlcNAc 2 Man 5 occur at the
cytosolic side of the ER membrane and use activated nucleotide sugars as donor
substrates. Synthesis of the oligosaccharide is completed at the luminal side of the
ER, but here cytosolically synthesized dolichol-linked membrane anchored mono-
saccharides serve as donor substrates (Figure 6.2, inset and Info Box 1 on evolu-
tionary aspects; please see also Figure 22.2 for pathway of LLO assembly) [3].
My six potential Asn sequons become glycosylated as soon as they are translo-
cated 40 Å (around 10 amino acids) into the ER lumen. The OST complex forms
together with other protein complexes a functional unit working on nascent poly-
peptide chains. Such tight positioning in close proximity to the entry pore of the
nascent polypeptide chain guarantees that OST acts on unfolded polypeptides with
their bendable Asn sequons. In most glycoproteins, this bending brings the chemi-
cally rather inert asparagine into close proximity with the OH groups of Thr/Ser,
or, in some proteins like von Willebrand factor and serum peptide C, with the SH
group of Cys. Note that proline inside or just after the Asn sequon prevents this
bending and prohibits glycosylation. Glycosylation effi cacy may decrease drasti-
cally along the last 70 C-terminal amino acids because of the accelerated transloca-
tion speed of the ribosome- released polypeptide [2] .
The mammalian OST complex is composed of a subset of different subunits
that perform the diverse enzymatic functions. Its STT3 subunits A or B recognize
Asn sequons and provide catalytic activity in concert with polypeptide-specifi c
subcomponents, such as ribophorin I. OST containing the ubiquitously expressed
mammalian STT3B (Q8TCJ2) exerts the basal glycosylation activity. STT3A
(P46977) is expressed in professional secretory cells such as hepatocytes in which
mature LLOs consumption is extremely high. SST3A containing OST strongly