Desialylation

As mentioned in Section 11.2, a special class of proteinaceous targeting constructs are those in which a therapeutic protein is used as the active drug substance. In such a preparation, the protein is redirected to the target tissue by the attachment of site-directing ligands such as those discussed in Section 11.3. For instance, interferon beta (IFN- 3) can be redirected to the liver by enz5matic desialylation in a procedure similar to that described earlier for fetuin (Section 11.3.1). The resultant asialo-IFN- 3 was found to have an in vivo anti-viral effect when tested in a hepatitis B model in athymic nude mice [54]. 

Fig. 4 Stereoselective binding study of verapamil to AGP and desialylated AGP using frontal analysis. Electropherograms of 50 /aM racemic verapamil solution (A), 50 /U.M verapamil solution in 50 jjM human al-acidic glycoprotein solution (B), and 50 /jM verapamil solution in 50 /jM asiaolo al-acidic glycoprotein solution (C). (Reprinted with permission from Ref. 54. Copyright 2001 Elsevier Science.)...

Application of sialidase inhibitors for medical use is still in a premature state. It is imaginable that inhibitors would be useful drugs in infections, caused by micro-organisms, that lead to extensive production of sialidase, for example, in gas edema.371 In the oral cavity, plaque formation and dental caries may be influenced by desialylation of salivary glycoconjugates,399 and bacterial sialidases may play a role therein. This process may be retarded by secretion of the inhibitor Neu2en5Ac in saliva at concentrations which, in some cases, were found to be close to the K value for sialidases.34... 

These studies demonstrating a protective effect of sialic acid residues on serum glycoproteins provide an explanation for earlier, conflicting observations about the biological effect of, for example, desialylated erythropoietin, which stimulates erythropoiesis only after direct application to bone-marrow cell-cultures, and not after injection into the blood stream.469 In the latter experiment, only the native, sialylated hormone was active. Rapid clearance and inactivation of follicle-stimulating hormone,470 or interferon,471 after treatment with sialidase may be explained by uptake into liver cells. 

Sialic acid seems to be involved not only in regulation of the lifetime of soluble, serum glycoproteins but also of mammalian blood-cells. It was observed by Woodruff and Gesner474 that desialylated lymphocytes are reversibly trapped in liver they recirculate to the blood stream after about 24 h. This phenomenon was confinned with Listeria-specific, mouse T lymphocytes, which accumulated in the liver for one day, in contrast to the control cells.60 Reappearance of these cells in the circulation after one day may be explained by re-sialylation of their membrane glycoconjugates. This time period is in the range observed for the turnover of sialic acid in cell membranes, lasting, for example, for 33 h in rat-liver hepatocytes.475... 

Desialylation of thrombocytes478 477 and erythrocytes also leads to their disappearance from circulation. Treatment of erythrocytes from different mammals, including man, with sialidase results in rapid clearance of these cells from the blood stream within a few hours, a phenomenon first observed by Perona and coworkers,478 and later investigated in various laboratories it was reviewed in Ref. 479. In man, the life-time of red blood-cells decreases from the normal 120 days to 2 h after sialidase treatment.4811... 

Complete desialylation of erythrocytes is not necessary for erythrocyte sequestration liberation of 15-20% of membrane sialic acids was found to be sufficient for a significant diminution of the life-time of erythrocytes.481,482 For an in vitro system with rat-peritoneal macrophages (see later), this threshold value was found to be only 10% when using soluble V. cholerae sialidase, but it was —30% when the erythrocytes were treated with the same enzyme immobilized on Sepharose.488... 

These, and other, mechanisms possibly involved in erythrophagocytosis will have to be elucidated in the future. Also shoidd be investigated the matter of whether antigenic sites (proteins or lipids), other than the desialylated glycoproteins and glycolipids having penulti-... 

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...

Determination of ohgosaccharides in the positive mode leads to the separation of sialic acid from the glycans, therefore the results reveal the masses of desialylated ohgosaccharides. 

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