Transferrin glycosylation

Abnormalities of the glycosylation of transferrin occur in the congenital disorders of glycosylation (Chapter 47) and in chronic alcohol abuse. Their detection by, for example, isoelectric focusing is used to help diagnose these conditions. 

The equivalent of the tryptic fragment of human transferrin receptor has been expressed in Chinese hamster ovary cells and its structure determined at a resolution of 0.32 nm (Lawrence et ah, 1999). The asymmetric unit of the crystals contains four transferrin receptor dimers. Interpretable electron density is found for the entire tryptic fragment except for Arg-121 at the amino terminus, and density is also seen for the first N-acetylglucosamine residue at each of the N-glycosylation sites. The transferrin receptor monomer is made up of three distinct domains, organized such that the dimer is butterfly shaped (Figure 5.10, Plate 7). The likely orientation of the dimer with respect to the plasma membrane has been assigned on the basis of the... 

Bacterial hosts are inappropriate choices for expression of proteins such as the blue copper proteins stellacyanin, laccase, and ceruloplasmin which are extensively glycosylated. In these cases, it may be necessary to employ tissue cultures of appropriate origin to obtain the native protein. In this regard, the amino-terminal half of human serum transferrin, which lacks carbohydrate, has been expressed in high yield in baby hamster kidney cells by Funk et al. [13], while the glycosylated carboxyl-terminus has proved to be more problematic [103]. 

CDT is the collective name of a gronp of minor isoforms of serum transferrin [14] with a low degree of glycosylation, inclnding asialo-, monosialo-, and disialo-Tf. CDT concentrations in serum are elevated during prolonged alcohol overconsnmption (>50-80 g/day for at least 7 days) and decrease, after... 

Final stage in the synthesis of a neura-minidate-containing carbohydrate unit of a glycoprotein (human transferrin). This terminal glycosylation takes place in the Golgi apparatus. 

A novel approach in initial CDG diagnostics has currently been described by analysing the glycosylation state of serum transferrin using electrospray ionisation-tandem mass spectrometry (ESI-MS/MS). This method requires expensive technical... 

Data are analysed by comparing the a-1-antitrypsin IEF patterns of patients suspected of having an altered transferrin protein backbone to healthy controls and patients with an already defined CDG. A normal pattern of a-1-antitrypsin in questionable patients indicates changes in the protein moiety of transferrin instead of a glycosylation deficiency. 

Using tunicamycin, Struck and coworkers203 demonstrated that, despite the inhibitory effect of the drug on glycosylation, secretion of transferrin, serum albumin, and apoproteins A and B of VLDL by primary cultures of rat and chick hepatocytes was virtually unimpaired. Keller and Sivank204 demonstrated the same point by showing that,... 

In the context of dmg delivery, certain cell surface receptor and ion-channel families are particularly appealing as dmg delivery targets. Well-characterized examples include the lectin-like receptor gene family as receptors for glycosylated molecules, as well as vitamin- and trace element-uptake systems such as the transferrin-receptor. 

Fig. 4. Ribbon diagram of human diferric lactoferrin, showing the organization of the molecule, with the N-lobe above and C-lobe below. The four domains (Nl, N2, Cl, C2), the interlobe connecting peptide (H), and the C-terminal helix (C) are indicated. The glycosylation sites in various transferrins are shown by triangles and numbered (1, human transferrin 2, rabbit transferrin 3, human lactoferrin 4, bovine lactoferrin 5 chicken ovotransferrin). The interdomain backbone strands in each lobe can be seen behind the iron atoms. Adapted from Baker et al. (82), with permission.

The recombinant whole molecules are both expressed in glycosylated form, although the glycosylation patterns differ from the proteins isolated from natural sources. The recombinant human transferrin binds to receptors both in its glycosylated form and as a nonglycosylated mutant, showing that the carbohydrate is not required for receptor binding (230). Recombinant human lactoferrin shows identical spectroscopic properties and shows an identical profile of pH-dependent iron release when compared with human milk lactoferrin (231). 

AO = Ascorbate oxidase (h)Cp = (human) Ceruloplasmin CT = Charge transfer Hp = Hephaestin GPl = Glycosyl-phosphatidylinositol Lac = Laccase MCO = Multicopper oxidase T1(2,3)D = Type 1 depleted (and/or type 2 or type 3) Tf = Transferrin. 

Chromatographic Measurement of Transferrin Glycoforms for Detecting Alcohol Abuse and Congenital Disorders of Glycosylation... 

Qualitative modifications in the glycosylation of this protein occur after prolonged heavy alcohol consumption and also in patients with rare congenital disorders of glycosylation (CDG). Therefore, measurement of transferrin microheterogeneity has been used both as a biomarker for detection and follow-up of alcohol abuse [known as carbohydrate-deficient transferrin (CDT)]" and for diagnosis of CDG. This chapter will review the microheterogeneity of human transferrin and the methods used for the measurement of transferrin... 

Figure 6.1 Structural illustration of the major normal glycoforms of human serum transferrin and those being influenced by chronic alcohol abuse (carbohydrate-deficient transferrin CDT) and in congenital disorders of glycosylation (CDG)...

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