Glycosyl residues sequencing

Glycosydation AChE and BChE carry 3 and 9, respectively, N-glycosylation consensus sequences attaching carbohydrate residues to the core protein via asparagines. Different molecular forms of the enzymes in various tissues, show different number and composition of carbohydrate residues. N-glycosylation at all sites was shown to be important for effective biosynthesis, secretion and clearance of ChEs from the circulation. Altered patterns of AChE glycosylation have been observed in the brain and cerebrospinal fluid of Alzheimer s disease (AD) patients, with potential diagnostic value. 

N-linked glycosylation is sequence specific, involving the transfer of a pre-synthesized oligosaccharide chain to an asn residue found in a characteristic sequence Asn-X-Ser, or Asn-X-Thr or Asn-X-Cys, where X represents any amino acid residue, with the exception of proline. An additional glycosylation determinant must also apply, as not all potential N-linked sites are glycosylated in some proteins. The pre-synthesized oligosaccharide side chain then undergoes... 

Especially noteworthy are the methods for determining the sequence of glycosyl residues in a complex oligo- or poly-saccharide. Methods of this type generate a series of overlapping oligomers that are separated and identified by l.c., in conjunction with either direct or indirect mass spectrometry. Most of these methods are still in the developmental stages, and they often require expensive and not-routinely available equipment (see also. Section IV,2) (see Addendum). 

At one linkage of this type in the chondroitin 4-sulfate from pig costal-cartilage,197 the sequence of amino acids is reported to be -Glu-Gly-Ser-Gly-, where Ser is the glycosylated residue. 

Various techniques have been utilized for sequencing the glycosyl residues of polysaccharides and oligosaccharides. These techniques are elaborations of the commonplace procedure of converting polysaccharides, by partial hydrolysis, into structurally analyzable oligosaccharides. This conversion is generally achieved by partial hydrolysis with acid, by acetolysis,131 or by formolysis.62 These reagents may differ in the... 

The main emphasis of current carbohydrate structural analysis is the applicability of modern multi-dimensional NMR for solving the two crucial problems in complex carbohydrate structural analysis, namely, the elucidation of the sequence of glycosyl residues and the solution conformation and dynamics of a carbohydrate (150). Techniques include 2D Total Correlation Spectroscopy (TOCSY), Nuclear Overhauser effect spectroscopy (NOESY), rotational nuclear Overhauser effect spectroscopy (ROES Y),hetero-nuclear single quantum coherence (HSQC), heteronuclear multiple quantum correlation (HMQC), heteronuclear multiple bond correlation (HMBC), and (pseudo) 3D and 4D extensions. 

In addition to determining the monosaccharide composition, linkage types, and sequence of residues in a cell wall polysaccharide, it is also essential to determine the anomeric configuration of the glycosyl residues. As with determining the sequence of glycosyl residues, the method of choice, be it chemical, enzymic, or physical, are dependent on the type of material under investigation and the amount available. 

---