Oligosaccharides transferrin

Fig. 4.5.9 HPLC and mass-spectromet-ric analysis of transferrin-linked oligosaccharides. Transferrin is purified from sera of a control and the index CDG-IId patient. Oligosaccharides are released by N-glycosidase F digestion and subsequently analysed by HPLC. Peak fractions of the control and the patient are further investigated by mass spectrometry and are compared to oligosaccharide standards. Values above the HPLC peaks indicate the detected masses...

Isoelectric focusing of transferrin is a useful biochemical test for assisting in the diagnosis of these conditions truncation of the oligosaccharide chains of this protein alters its isolectric focusing pattern... 

Fig. 4.5.2 Actual strategies for CDG diagnosis. Initial investigations on CDG patients are routinely carried out by isoelectric focusing (IEF) of serum transferrin. With a CDG type I pattern, subsequent analysis should imply determination of phosphomannomutase (PMM) and phos-phomannose isomerase (PMI) activities. Further studies, like analysis of the lipid-linked- and protein-bound-oligosaccharides, determination of enzyme or sugar transporter activities and molecular biology studies often have to be performed in more specialised laboratories. HPLC High-performance liquid chromatography, TLC thin-layer chromatography...

In all known types of CDG-I it has been observed that due to the loss of sialic acid residues, in addition to tetrasialo-transferrin, more or less pronounced di- and asialo-transferrin bands appear, which are evoked by the loss of either one or both complete oligosaccharide chains (Fig. 4.5.3, lanes 2-4). In contrast to CDG-I, changes in the charge of serum transferrin of most known CDG-II types are due to shortening of the oligosaccharide moieties (Fig. 4.5.3, lanes 5-7). 

Fig. 4.5.4 Identification of mutations in the transferrin protein by neuraminidase treatment. Unusual patterns in the IEF of serum transferrin might lead to pitfalls in CDG diagnostics. These varying patterns are often due to mutations of charged amino acids in the protein backbone of the transferrin molecule, which might lead, for example, to an accumulation of trisialo transferrin bands (lane 3, indicated by a question mark). Further investigations are carried out by cleaving off charged sialic acid monosaccharide moieties from transferrin-linked oligosaccharides by neuraminidase treatment, followed by IEF and transferrin antibody staining. In the case of protein mutations, additional bands below (lane 4) or above (not shown) the desialylated transferrin form appear...

Release of Oligosaccharides from Purified Transferrin by N-Glycosidase F Digestion... 

Fig. 4.5.8 Examples for structures and molecular masses of 2-aminobenzamide (2-AB)-labelled oligosaccharide moieties derived from serum transferrin. Values below the oligosaccharide structures indicate the expected masses (in Da) by matrix-assisted laser desorption ionisation - time of flight analysis.

Bayard investigated a number of glycoproteins (aracid glycoprotein, fetuin, lactotransferrin, transferrin, and ovomucoid) by N-deacetylation followed by deamination.176 They all gave the oligosaccharides 108 and 109, demonstrating the presence of common structural elements in these compounds. 

Figure 9.11 Structure of an oligosaccharide unit of human transferrin. The linkage between the oligosaccharide and polypeptide chains is of the N-asparaginyl type. Dotted arrows indicate positions in which one sometimes finds NeuNAc-Gal-GlcNAc units. Gal is galactose, GIcNAc is N-acetylglucosamine, NeuNAc is N-acetylneuraminic acid (sialic acid), and Man is mannose. (Reproduced with permission from Bezkorovainy A. Biochemistry of Nonheme Iron. New York Plenum Press, 1980, p. 175.)...

Oligosaccharide units, discussed in the previous section, are relatively small. The molecular weight of the transferrin oligosaccharide group, for example, is... 

Other molecules have been suggested as being useful lung-specific bioadhesive agents [137,138], for example, insulin, transferrin, prostaglandins, hirudin-inhibited thrombin (which binds thrombomodulin), anionic polysaccharides, oligosaccharides (such as dextran sulfate, dermatan sulfate, chondroitin sulfate, hyaluronic acid), peptides (such as benzoyl-phe-ala-pro [BPAPI] that... 

The function and structure of the oligosaccharides on the iron-binding transferrin family of proteins is dealt with in detail in Chapter 10. 

CDG-type I syndrome [264,266,267] is due to a deficiency in the oligosaccharidyltrans-ferase which transfers en bloc onto the nascent protein the oligosaccharide linked to dolichol diphosphate. Later, van Schaftingen and Jaeken [1995, FEES Lett. 377, 318-320] demonstrated that the syndrome was due in fact to a phosphomannomutase deficiency, an enzyme which provides the mannose-1-phosphate required for the initial steps of protein glycosylation. This leads to four transferrin isoforms non-glycosylated, glycosylated in Asn-413 or in Asn-611 and in both Asn-413 and 611. 

Chemical analysis throws a little light on the problem. Oligosaccharides are known from several sources in which the bi-antennary structure of asialo-transferrin occurs as the inner part of sialo-glycopeptide sequences. In some the structure has two terminal a2,3 sialyl groups (Strecker etal, 1977 Burke and Keegstra, 1979), while in others both are in the a2,6 configuration. Cases of simultaneous presence of both types of link are also known (Strecker etal, 1977). 

Oligosaccharides from glycopeptides by hydarazinolyses Derivatization 2-pyridylamine dimethylamine/borane D FI Aex = 320 nm Aem = 380 nm C Fused silica (60 cm x 50 pm i.d.) polyimine coated E 100 mmol 1 phosphate, pH 2.5 200 mmol I borate V 20 kV Glycopeptides obtained by trypsine digestion of human transferrin, serum fetuin, human immunoglobulin, bovine, spleen ribonuclease, yeast invertase... 

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