Primary structure glycosylation sites

Fig. 2. Primary structure of hamster PrP (Stahl et al., 1993). The first 22 residues at the N-terminus are the signal sequence. PrPc is completely digested by proteinase K, whereas the N-terminal sequence of PrPSc to residue 89 (arrow, closed head) is digested. — CHO indicates the glycosylation sites at residues 181 and 197 Gpi the glycosylpho-sphatidylinositol anchor at 231 and the N-terminal octarepeats. In one case of human prion disease, a stop codon was found at 145 (arrow, open head) (Kitamoto et al., 1993). HI, H2, H3, and H4 denote the predicted a-helices (Huang et al, 1994, 1996), and A-C denote the a-helices and SI, S2 the /(-strands determined by solution NMR (James et al, 1997).

Fig. 5.6. Topology of the P-adrenergic receptor of hamster. The primary structure is shown of the P-receptor for adrenaline from hamster, with the assumed topology of the seven transmembrane helices. The extracellular domain is shown at the top of the picture. The interface of the ceU membrane is indicated by the dashed line. The filled squares show glycosylation sites. Amino adds not required for ligand binding, according to mutagenesis studies, are shown as open squares. Reprinted with permission of the American Journal of Respiratory Cell and Molecular Biology (1989), 1, No.2, p.82.

Antithrombin is a member of the SERPIN superfamily of proteins, which includes the inhibitors a2 an1 Pbsniin, ar antichymotrypsin, and a -proteinase inhibitor (79). Antithrombin is considered to be the primary inhibitor of coagulation (80) and targets most coagulation proteases as well as the enzymes trypsin, plasmin, and kallikrein (81). Inhibition takes place when a stoichiometric complex between the active site serine of the protease and the ARG393-SER394 bond of antithrombin forms (82,83), The tertiary structure of antithrombin resembles a,-antitrypsin in that it is folded into N-terminal domain helices and (3-sheets. This tertiary structure is maintained by the formation of three disulfide bonds (71). Four glycosylation sites exist on human... 

A further consideration is the position of the sequon in relation to the primary structure of the protein. Statistical analysis of a large number of glycoproteins has indicated that the frequency of non-glycosylated sequons increases toward the C-terminus (Gavel and von Heijne, 1990). The critical distance appears to be 60 amino acid residues from the C-terminus when reduced glycan occupation occurs. This distance corresponds to the distance between the ribosome P-site and the active site of the OST and it has been hypothesized that the protein chain is not available for N-glycan attachment once it is released from the ribosome. However, this phenomenon of poor glycosylation efficiency toward the C-terminus does not appear to be universal for all proteins (Walmsley and Hooper, 2003). 

Peptide Mapping. Peptide mapping is an important tool for protein identif-ication, primary structure determination, the detection of posttranslational modifications, the identification of genetic variants, and the determination of glycosylation and/or disulfide sites. For these reasons, peptide mapping is widely used for quality control and for the characterization of recombinant DNA-derived products. Moreover, the high resolution of CE makes it a powerful peptide mapping technique. 

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