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30% of consensus sequences for N-linked attachments are occupied by polysacchar-

ide; the nature of the secondary structure at this position also seems to play a role

in deciding whether glycosylation takes place), variations in the type of amino

acid–carbohydrate bond, variations in the composition of the sugar chains, and

variations in the particular carbohydrate sequences and linkages in each chain. There

are eight monosaccharide units commonly found in mammalian glycoproteins,

although other less common units are also known to occur. These eight areN-acetyl

neuraminic acid (NeuNAc),N-glycolyl neuraminic acid (NeuGc),D-galactose (Gal),

N-acetyl-D-glucosamine (GlcNac),N-acetyl-D-galactosamine (GalNAc),D-mannose (Man),

L-fucose (Fuc) andD-xylose (Xyl). To further complicate the issue, within any population

of molecules in a purified glycoprotein there can be considerable heterogeneity in

the carbohydrate structure (glycoforms). This can include some molecules showing

increased branching of sugar side-chains, reduced chain length and further addition of

single carbohydrate units to the same polypeptide chain. The complete determination

of the glycosylation status of a molecule clearly requires considerable effort. However,

the steps involved are fairly straight forward and the following therefore provides

a generalised (and idealised) description of the overall procedures used.

CH 2 OH

O

NH

CH

NH C

O

Ser orThr

O

C

CH 3

X

O CH

NH

C

Ser

(orThr)

GlcNAc

N-glycosylation

O

OH

CH 2

GalNAc

O-glycosylation

NH C Asn

O

CH 2

CH 2 OH

NH C

O

O

CH 3

O

OH

O

Fig. 8.3The two types of oligosaccharide linkages found in glycoproteins.

335 8.4 Protein structure determination