section 16.1
Biosynthesis of Glycoproteins
315
The secondary or tertiary structure can prevent glyco-
sylation of sites within those regions, and disruption of
tertiary structure allows glycosylation to occur at new sites
in certain glycoproteins. Availability of active transferase
and of lipid-linked oligosaccharide also affects the rate of
glycosylation.
(2)
Processing
rapidly follows transfer of the oligosac-
charide to the polypeptide. Stepwise removal of the
glucose residues and of some of the a (1
->■
2)-linked
mannose residues drastically alters the structure. The gly-
cosidases are located in the rough endoplasmic reticulum
and in the cis and medial portions of the Golgi apparatus
(Figure 16-7). Processing begins in the rough endoplasmic
reticulum with removal of three glucose residues and one
mannose residue. The partially processed, high-mannose
glycoprotein is then transferred to the cis Golgi region
closest to the endoplasmic reticulum, probably by bud-
ding of vesicles from the rough endoplasmic reticulum
that move to and fuse with the Golgi membrane. In
the cis
Golgi cistemae, high-mannose glycoproteins
destined for inclusion in lysosomes are phosphorylated,
while
other glycoproteins
are
further processed by
removal of three more mannose residues and transferred
to the medial Golgi region. This transfer is probably
mediated also by budding of vesicles from the cis Golgi
membrane.
(3)
Elongation
begins in the medial Golgi by addition
of GlcNAc to the 3'-core mannose, followed by removal
of the two mannose residues on the 6'-mannose branch.
Further elongation to form a complex-type oligosaccha-
ride proceeds in the medial and trans Golgi regions with
addition of GlcNAc, Gal, Fuc, and sialic acid. Transfer
from medial to trans Golgi and transport of completed
glycoproteins to elsewhere in the cell probably, occur by
vesicular transport. Many glycosyltransferases that cat-
alyze elongation reactions require an acceptor having the
correct terminal sugar and correct sugars at specific lo-
cations in the oligosaccharide. For example, the enzyme
that transfers fucose from GDP-fucose to the innermost
GlcNAc of the core oligosaccharide cannot function un-
less a GlcNAc has first been attached to the 3'-mannose at
the outermost end of the structure.
The final oligosaccharide product of this elongation pro-
cess is significantly influenced by the types and amounts of
glycosyltransferases present in the medial and trans Golgi.
Activation of
Ras
genes, associated with the metastatic
spread of tumors, have also been shown to increase the
expression of GlcNAcTransferase V. This enzyme may
play a role in cell motility and therefore its correlation
with tumor metastasis is significant, since motility is an
important factor in the initiation of metastasis.
Phosphorylation of Oligosaccharide
Chains on Lysosomal Enzymes
Some Asn-linked glycoproteins (e.g., the acid hydro-
lases destined for incorporation into lysosomes) undergo
phosphorylation of oligosaccharide chains. The phosphate
groups are attached to two penultimate mannose residues
on incompletely processed high-mannose side chains. The
first step is transfer of GlcNAc-1-phosphate to the mannose
acceptor sites, catalyzed by UDP-GlcNAc:lysosomal en-
zyme GlcNAc-1-phosphotransferase, to form a phospho-
diester linkage. This phosphotransferase is specific for
the Man
a(l -*■
2) Man linkage characteristic of high-
mannose structure and for an amino acid sequence in
the lysosomal protein. The GlcNAc is then removed by
an a-GlcNAc-phosphodiesterase, leaving a phosphoman-
nose residue (Figure 16-9). Several potential phosphory-
lation sites exist on a high-mannose oligosaccharide and
multiple isomers can occur.
Phosphate group transfer is important because the phos-
phomannosyl residues on these enzymes act as recognition
markers for their proper routing to lysosomes. Two hu-
man lysosomal storage disorders (Chapter 11) are caused
by a genetically determined reduction in the activity of
GlcNAc-phosphotransferase. In inclusion cell disease (/-
cell disease,
mucolipidosis II), the activity is completely
absent, whereas in pseudo-Hurler’s polydystrophy (mu-
colipidosis III), the activity is severely reduced. Both dis-
eases are characterized by high concentrations of certain
lysosomal enzymes in the plasma and by greatly reduced
activities of these and other acid hydrolases in lysosomes.
The plasma lysosomal enzymes in these disorders prob-
ably represent a particularly stable subset of misdirected
enzymes. Some have a higher molecular weight and more
negative charge than the corresponding enzymes in normal
lysosomes. The increased size may occur because the en-
zymes have not undergone the limited proteolysis that nor-
mally occurs within the lysosomes. The increased negative
charge is due to the presence of sialic acid residues added
during further processing and elongation of the nonphos-
phorylated proteins. Acid phosphatase and /f-glucosidase
occur in normal amounts in the lysosomes, suggesting that
a different mechanism directs them to the lysosomes.
Inhibitors of Glycoprotein Biosynthesis
The oligosaccharide chains on some glycoproteins may be
needed for receptor recognition, antigenicity, intracellular
transport and secretion, protection against proteolytic di-
gestion, and stability at extremes of temperature and pH. In
other glycoproteins, however, absence of the carbohydrate
