The paper describes the procedure of efficient elution of glycoproteins from Immobilon membrane after their blotting from polyacrylamide gel. The yield of elution from membrane is 30-70%.
The diphenylketone Shiff - base appeared to be a very good protection for amino groups of serine and threonine for glycosylation reaction. O-glycosides of these aminoacids with different monosaccharides, aminosugars and deoxysugars were obtained with excelent yield and very high stereoselectivity. Products of glycosylation reactions were used as building blocks for solid phase glycopeptide synthesis.
Baculovirus system has been used for the expression of genes of herpesvirus glycoproteins. In this system highly glycosylated proteins are obtained. The glycoproteins may find potential use as vaccines and in diagnosis of pseudorabies.
Many yeasts secrete proteins which are toxic for pathogenic and non-pathogenic microorganisms. These toxins, mostly glycoproteins, consist of membrane-binding subunits which interact with carbohydrates (e.g. 1,6--D-glucan or -mannan) on the cell wall of sensitive strains. The killing effect is presented by membrane permeation, cell lysis or inhibition of the cell cycle. It is also suggested that these killer glycoproteins, similar in structure to lectins, can mediate self-adhesion of the pathogenic microorganisms, thus stimulating their excretion from the intestines of infected mammals. It is supposed that the above interactions could be important for therapeutic applications, especially for enteric diseases. In order to fully understand the structural basis of the functions of killer glycoproteins, it is essential to characterize their glycosylation state and to determine the structure of all glycans attached to the proteins. In this paper, a strategic approach to the purification of yeast protein from complex biological mixtures is presented. The approach is structured into seven subassignments, each of which is essential for the successful isolation of a pure and biologically active yeast protein. The subassignments are: 1) decision on the use of the purified protein; 2) collecting information about the chemical, physical and biological properties of the protein; 3) establishing assays for the protein and its biological activity; 4) decision on the source of raw material; 5) development of an efficient extraction method; 6) development of a purification method; 7) establishment of optimum conditions for storage of the purified protein.
In this paper the occurrence and relative content of defectively glycosylated serum glycoforms in transferrin (Tf), 1-acid glycoprotein (AGP), haptoglobin (Hp), 1-antitrypsin (1-AT), 2-macroglobulin (2-MG) and ceruloplasmin (Cpl) in the serum of a patient with congenital disorder of glycosylation type I are reported. Blood samples were taken when the patient was 14 years old and then after a one-year interval. The patterns of glycoforms in both samples were compared. In 4 out of 6 examined glycoproteins, glycoforms lacking one and two oligosaccharide chains occurred. ?Underglycosylated? glycoforms of 2-MG and Cpl were not clearly detectable. Tf was shown to be affected with this defect to a higher extent than other glycoproteins, containing only 30% properly glycosylated molecules and also as much as 30% of the molecules lacking two glycan units. In Hp and 1-AT the proportions of properly and defectively glycosylated forms were similar. This properly glycosylated form comprised 47% of the Hp and 51?55% of the 1-AT molecules. As in AGP and Tf, about 30% the of molecules lacked one glycan unit. Twenty-one percent of the Hp molecules were devoid of two glycans, and this amount slightly increased in the course of the year. In 1-AT, 19 and 17% of the molecules lacked two glycans in both samples, respectively. Only in AGP we did find a substantial difference between the two blood samples. In the course of the year, the amount of the form lacking 2-chains decreased from 12 to 3%, resulting in a simultaneous increase in the forms lacking one chain and the properly glycosylated. Our work also indicates, that applying a simple method of biochemical analysis such as SDS-PAGE/Western-blotting could be helpful in preliminary diagnosis and could improve the identification of congenital disorders of glycosylation.
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