Protein Glycosylation: Implications for In Vivo Functions and Therapeutic Applications - Advances in Biochemical Engineering Biotechnology - page 186

semisynthetic methods for the generation of lead molecules [251]. This opens
the possibility of adding defined glycan structures after recombinant protein
synthesis and secretion. In addition, cloning and expression of several impor-
tant glycosyltransferases in E. coli will allow post-harvest remodeling of glyco-
proteins produced in cell culture,making them more acceptable as human thera-
peutic proteins [252].
8
Characterization of Sugars
Assessment and control of product heterogeneity are the major problems faced
in the manufacture of recombinant proteins. Unraveling the complexities of
glycosylation of a molecule is a substantial task. Sequence analysis has a some-
what more entangled meaning for a variably linked and multiple branched
structure than for the linear biopolymer as in the case of peptides. Deglycosyla-
tion reactions may be discriminating or incomplete and chromatography could
enrich or exclude particular subforms. However, in many situations this is not
necessary and only a limited amount of information on a single or a group of
structural features is needed. Techniques and instruments are now becoming
available which, particularly when used in combination, can provide rapid and
accurate comparisons of the glycosylation patterns of glycoproteins.
Glycan analysis is performed on intact glycopeptide or after their release
from the protein. Thus, two approaches generally practiced to compare the
glycosylation pattern of glycoproteins are (a) glycan analysis on intact glyco-
peptide - site-specific digestion of glycoprotein with proteases or cynogen
bromide to yield smaller glycopeptides, separating them, and analysing amino
acid sequence and glycan structure by different standard techniques, and (b)
glycan analysis - releasing the individual glycans from the glycopeptide or intact
protein by chemical or enzymatic treatment, labeling and separating them, and
determining the relative quantity of individual released saccharide. Details of
procedural aspects for the analysis of glycans are beyond the scope of this
review, although a brief description of alternative methods for the analysis and
their scope are presented in Table 4. Excellent reviews pertaining to the structu-
ral analysis of glycans have been published recently [228, 268].
9
Conclusions
This review has addressed the glycosylation of proteins, its role in biological
functions,associated diseases due to defects in protein glycosylation,expression
systems available for the production of recombinant glycoproteins and their
characterization.Although much progress has been made towards understand-
ing the biological roles of glycosylation and consequences of altered co-/post-
translational cellular events, the areas that need to be attended to are the mole-
cular basis of altered glycosylation, and control of glycosylation in the branch