Glycosylation of Model and ‘Lower’ Organisms - The Sugar Code - page 141

pathogen), of glucose to tyrosine in the S-layer glycoprotein of Thermoanaerobac-
terium thermosaccharolyticum (an anaerobic thermophile), of O - linked GlcNAc to
fl agellin in Listeria monocytogenes (a food-borne pathogen) and of O - linked mannose
to proteins of mycobacteria (including the bacterium responsible for
tuberculosis).
8.2
Yeast Glycosylation
The simplest eukaryotic systems are obviously unicellular organisms and, amongst
these, the use of the baker's yeast Saccharomyces ( S.) cerevisiae as a glycobiological
model has a relatively long tradition. The pioneering work of Phillips Robbins in
the 1980s in the use of yeast to isolate alg (asparagine - linked glycosylation) mutants
with defects in the biosynthesis of the dolichol-linked intermediates for N - glycans
was important in laying the foundations for our present knowledge about the
genetic basis for this pathway in eukaryotes [2]. Today, we know which genes are
necessary for each enzymatic step leading to the generation of the dolichol- linked
form of Glc 3 Man 9 GlcNAc 2 , which serves as the oligosaccharyltransferase substrate
in most eukaryotic species (see Chapter 6 on N - glycosylation and Chapter 22 on
aberrations in the biosynthetic pathway). The ability to generate yeast mutants and
complement them with isolated yeast or human genes has been key for this work.
Furthermore, these studies have aided uncovering the genetic basis for a number
of the human congenital disorders of glycosylation listed in Table 22.1 .
It would, though, be a mistake to think of yeast as just meaning S. cerevisiae ;
this is particularly clear when one considers the modifi cations of the N - glycans
once they pass through the Golgi apparatus. S. cerevisiae has the tendency to
hyper mannosylate its N -glycans; unlike mammals, this yeast has, in addition to
those conserved mannosyltransferases in the endoplasmic reticulum ( ER ) required
for the biosynthesis of the dolichol-linked precursor, many Golgi-localised man-
nosyltransferases, which utilise GDP-Man as a donor substrate [3] . Tens of
mannose units (some of which are phosphorylated) may be added, resulting in
very large N -glycans on some glycoproteins (see Figure 8.2), particularly those of
the cell wall. This has the repercussion that recombinant glycoproteins expressed
in S. cerevisiae also tend to be hypermannosylated, which may negatively affect
their physicochemical, biological or enzymatic properties. However, due to their
ability to produce high amounts of recombinant proteins, yeasts are biotechnologi-
cally interesting; the result is that a number of other yeast species have been
examined as potential 'cell factories'. For instance, methylotrophic yeasts such as
Pichia pastoris or Ogataea minuta are used as expression systems. They tend to add
fewer mannose residues to their N -glycans (structures as large as Man 15 GlcNAc 2
are typical, instead of the sometimes 100 mannose units in some S. cerevisiae
glycans); these are still not akin to the glycans found in mammals. Thus, the ability
to also easily knock-out genes in yeasts has been exploited to re- engineer glycosyl-
ation in these species. Particularly, the och1 (outer chain 1) genes, which encode