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chapter 16
Carbohydrate Metabolism III: Glycoproteins, Glycoiipids, GPI Anchors, Proteoglycans, and Peptidoglycans
3UDP-<
4 GOP
2UDP
Manal— *
Manal
^ManaK^
—>2ManaK^
dolichol phosphate
♦
-
N-acetylglucosamme
glucosamine
# -
mannose
► -
glucose
C D
protein
P p o
phosphatidylinositol
*Mano1
Glcol-
*2Glca1— 3Glco1— * 3 Manal— *2Mana1— 2M ano1^*
®Man pi— 4GlcNAc p1— »4GtcNAc-P-P-dolichol
F I G U R E 1 6 -8
Topography of glycosylation in the rough endoplasmic reticulum. Initiation of N-glycosylation and transient
reglucosylation. [Reproduced with permission from C. Abeijon and C. B. Hirschberg, Topography of glycosylation
reactions in the endoplasmic reticulum.
T rends B iol. Sci.
17:34 (1992).]
lumen from dolichol-P-mannose and possibly dolichol-
P-glucose donor substrates that were synthesized on the
cytoplasmic side of the membrane and then translocated to
the lumen. The dolichol-P-mannose transfer to the mem-
brane lumen is essential since GDP-mannose cannot be
transported across the endoplasmic reticulum. Both UDP-
GlcNAc and UDP-Glc can be transported across the mem-
brane. The final lipid-linked oligosaccharide is then ready
for transfer to nacsent polypeptides that extend into the cis-
ternae from the cytosolic surface to the rough endoplasmic
reticulum.
Transfer of the oligosaccharide to an asparagine residue
on a polypeptide occurs in the cisternae of the rough
endoplasmic reticulum (Figure 16-8), during or soon
after the synthesis of the protein. The oligosaccharyl-
transferase is a large heteroligomeric membrane protein
complex that shows significant conservation of sequence
between
vertebrate,
invertebrate,
plant,
and
fungal
subunits. The transferase is specific for the sequence N-
X-T/S [Asn-X-Ser(Thr)], in which X can be any residue
except prolyl or aspartyl. Proline at the +3 position also
prevents transfer. The N-linked carbohydrates have also
been shown to be attached to N-X-C sequences for human
protein C and von Willebrand factor. It has been pro-
posed that the seryl (or threonyl) hydroxyl in the triplet
participates in catalysis by activation of the amide nitro-
gen of asparagine for reaction with the donor substrate,
oligosaccharide-dolichol. Statistical analysis of glycosy-
lated consensus sites in glycoproteins showed that N-X-T
sites were glycosylated about twice as often as N-X-S
sequons. Experiments using peptides showed a 40-fold
preference for N-X-T over sequences containing serine.
In addition, glycosylation sites located toward the COOH
terminal end of proteins were less frequently glycosy-
lated. Clearly, additional mechanisms must play a role in
N-glycosylation.
