Biochemical Society Transactions ( 1 992) 20 325s
Comparison between membrane sialoglycoproteins present in rat and human erythrocytes ANGEL HERRAEZ, JOSE C. DIEZ and JOSE LUQUE. Dpto. Bioqufmica y Biologfa Molecular, Universidad de Alcali 28871 Alcali de Henam, Madrid, Spain Erythrocyte membranes contain a major group of sialoglycoproteins, named glycophorins. They are heavily glycosylated intrinsic membrane proteins which have been claimed to be restricted to erythroid lineage, where they may be implicated in a variety of functions [l-41. Among the roles proposed for glycophorins are the interaction of viruses and bacteria with the erythrocyte and their implication in processes involving cell morphological changes. In humans, glycophorins have been extensively characterized. Structural epitopes determining blood groups MN and Ss are located on human glycophorins. Related sialoglycoproteins have also been isolated from erythrocytes of other species (see [5] for a review). Structural comparison among glycophorins from different species may shed light on the general roles performed by these glycoproteins as well as on the specific functions performed in each case. In rat, two isolation methods have been successfully applied [6,7 rendering in both cases a population of four sialoglycoproteins on SDS-PAGE stainable by periodic acidSchiffs reagent. The estimated molecular weight of these rat glycophorins are 74, 32, 25 and 17 kDa as inferred from electrophoretic analyses [7]. Comparison of human and rat glycophorins shows different electrophoretic patterns, although some bands with analogous molecular weights appear in both species (Fig. 1). Application of the isolation procedure involving the use of the detergent lithium diiodosalicylate [8,9] to human and rat erythrocyte membranes gives preparations showing different electrophoretic patterns, although some bands with analogous molecular weights appear in both species (Fig. 1). Quantitative analysis renders a similar result for sialic acid to protein ratio in both purified glycophorins: 0.35 and 0.41 (w/w) for rat and human, respectively. Yields were also similar in both cases, accounting for 32% and 35%of the sialic acid in the membrane, and 1.5% and 1.8% of its total protein (in rat and human respectively). Determination of the levels of 0-acetylsialic acids in rat and human glycophorins revealed the presence of this molecular variety in rat glycophorins only [10,11]. The amount of 0-acetylated sialic acids in rat glycophorins was one third of the total sialic acid (i.e., N-acetylneuraminic plus O-acetylN-acetylneuraminic acids), therefore the above-mentioned sialic to protein ratio must be corrected up to 0.52 for this species. Additionally, preparation of antibodies against rat glycophorins enabled to study the relationship with their human counterparts. To this purpose, both an antiserum against the whole preparation of rat erythrocyte glycophorins and an antiserum against the 32 kDa component were tested on dot-blot analyses with human and rat glycophorins. At sufficiently high levels of antigen dotted, both antisera show crossrreaction with human glycophorins, though rather weaker than the reaction with rat glycophorins. Hence, some structural relationship between rat and human glycophorins may be inferred. On the other hand, monoclonal anti-human glycophorins antibody (MC-26, Sigma Chemical Co.) did not recognize rat glycophorins in dot-blot experiments. Thus, the absence in the rat sialoglycoproteins of a region of human glycophorins is
kDa
-
116 68
45
29
-
-
- 18
Figure 1: ElectroDhoretic profiles of rat (left) and human a h t ) ~urifiedelvcoDhoring. Samples were run on 0.1 % SDS/11% polyacrylamide gels and stained with periodic acid-Schiff s reagent.
confirmed. The evidence of homologies and differences between rat and human glycophorins may be an important clue in the understanding of the functional roles performed by these sialoglycoproteins in different species. So, analogous regions might correspond to common functions in different species, while structural discrepancies could justify the involvement of glycophorins in specific functions in different species. We acknowledge finantial support from C.1.C.Y T., Comunidad Aut6noma de Madrid and the University of Alcali de Henares. 1. Marchesi, V.T., Furthmayr, H. & Tomita, M. (1976) Annu. Rev. Biochem. &5, 667-698 2. Furthmayr, H. (1978) J. Supramol. Struct. 9, 79-95 3. Gahmberg, C.G., Jokinen, M. & Andersson, L.C. (1978) Blood 2, 379-387 4. Bizot, M. (1990) Dictionnaire des antigenes erythrocytaires immunogenes. pp. 82-85 and 21 1-230. Sauramps M&ical, Montpellier 5 . Krotkiewski, H. (1988) Glycoconjugate J. 5, 35-48 6. Edge, A.S.B. & Weber, P. (1981) Arch. Biochem. Biophys. 209, 697-705 7. Hendez, A., Diez, J.C. & Luque, J. (1992) Int. J. Biochem. in the press 252-254 8. Marchesi, V.T. (1972) Methods Enzymol. 3, 9. Marchesi, V.T. & Andrews, E.P. (1971) Science 174, 1247-1248 10. Sarris, A.H. & Palade, G.E. (1979) J. Biol. Chem. 254, 6724-673 1 11. Reuter, G., Vliegenthart, J.F.G., Wember, M., Schauer, R. and Howard, R.J. (1980) Biochem. Biophys. Res. Commun. 94,567-572
Comparison between membrane sialoglycoproteins present in rat and human erythrocytes ANGEL HERRAEZ, JOSE C. DIEZ and JOSE LUQUE. Dpto. Bioqufmica y Biologfa Molecular, Universidad de Alcali 28871 Alcali de Henam, Madrid, Spain Erythrocyte membranes contain a major group of sialoglycoproteins, named glycophorins. They are heavily glycosylated intrinsic membrane proteins which have been claimed to be restricted to erythroid lineage, where they may be implicated in a variety of functions [l-41. Among the roles proposed for glycophorins are the interaction of viruses and bacteria with the erythrocyte and their implication in processes involving cell morphological changes. In humans, glycophorins have been extensively characterized. Structural epitopes determining blood groups MN and Ss are located on human glycophorins. Related sialoglycoproteins have also been isolated from erythrocytes of other species (see [5] for a review). Structural comparison among glycophorins from different species may shed light on the general roles performed by these glycoproteins as well as on the specific functions performed in each case. In rat, two isolation methods have been successfully applied [6,7 rendering in both cases a population of four sialoglycoproteins on SDS-PAGE stainable by periodic acidSchiffs reagent. The estimated molecular weight of these rat glycophorins are 74, 32, 25 and 17 kDa as inferred from electrophoretic analyses [7]. Comparison of human and rat glycophorins shows different electrophoretic patterns, although some bands with analogous molecular weights appear in both species (Fig. 1). Application of the isolation procedure involving the use of the detergent lithium diiodosalicylate [8,9] to human and rat erythrocyte membranes gives preparations showing different electrophoretic patterns, although some bands with analogous molecular weights appear in both species (Fig. 1). Quantitative analysis renders a similar result for sialic acid to protein ratio in both purified glycophorins: 0.35 and 0.41 (w/w) for rat and human, respectively. Yields were also similar in both cases, accounting for 32% and 35%of the sialic acid in the membrane, and 1.5% and 1.8% of its total protein (in rat and human respectively). Determination of the levels of 0-acetylsialic acids in rat and human glycophorins revealed the presence of this molecular variety in rat glycophorins only [10,11]. The amount of 0-acetylated sialic acids in rat glycophorins was one third of the total sialic acid (i.e., N-acetylneuraminic plus O-acetylN-acetylneuraminic acids), therefore the above-mentioned sialic to protein ratio must be corrected up to 0.52 for this species. Additionally, preparation of antibodies against rat glycophorins enabled to study the relationship with their human counterparts. To this purpose, both an antiserum against the whole preparation of rat erythrocyte glycophorins and an antiserum against the 32 kDa component were tested on dot-blot analyses with human and rat glycophorins. At sufficiently high levels of antigen dotted, both antisera show crossrreaction with human glycophorins, though rather weaker than the reaction with rat glycophorins. Hence, some structural relationship between rat and human glycophorins may be inferred. On the other hand, monoclonal anti-human glycophorins antibody (MC-26, Sigma Chemical Co.) did not recognize rat glycophorins in dot-blot experiments. Thus, the absence in the rat sialoglycoproteins of a region of human glycophorins is
kDa
-
116 68
45
29
-
-
- 18
Figure 1: ElectroDhoretic profiles of rat (left) and human a h t ) ~urifiedelvcoDhoring. Samples were run on 0.1 % SDS/11% polyacrylamide gels and stained with periodic acid-Schiff s reagent.
confirmed. The evidence of homologies and differences between rat and human glycophorins may be an important clue in the understanding of the functional roles performed by these sialoglycoproteins in different species. So, analogous regions might correspond to common functions in different species, while structural discrepancies could justify the involvement of glycophorins in specific functions in different species. We acknowledge finantial support from C.1.C.Y T., Comunidad Aut6noma de Madrid and the University of Alcali de Henares. 1. Marchesi, V.T., Furthmayr, H. & Tomita, M. (1976) Annu. Rev. Biochem. &5, 667-698 2. Furthmayr, H. (1978) J. Supramol. Struct. 9, 79-95 3. Gahmberg, C.G., Jokinen, M. & Andersson, L.C. (1978) Blood 2, 379-387 4. Bizot, M. (1990) Dictionnaire des antigenes erythrocytaires immunogenes. pp. 82-85 and 21 1-230. Sauramps M&ical, Montpellier 5 . Krotkiewski, H. (1988) Glycoconjugate J. 5, 35-48 6. Edge, A.S.B. & Weber, P. (1981) Arch. Biochem. Biophys. 209, 697-705 7. Hendez, A., Diez, J.C. & Luque, J. (1992) Int. J. Biochem. in the press 252-254 8. Marchesi, V.T. (1972) Methods Enzymol. 3, 9. Marchesi, V.T. & Andrews, E.P. (1971) Science 174, 1247-1248 10. Sarris, A.H. & Palade, G.E. (1979) J. Biol. Chem. 254, 6724-673 1 11. Reuter, G., Vliegenthart, J.F.G., Wember, M., Schauer, R. and Howard, R.J. (1980) Biochem. Biophys. Res. Commun. 94,567-572
