The erythrocyte cytoskeleton protein 4 . 2 is not demonstrable in several mammalian species

Erythrocyte membrane proteins from 44 representative mammals were studied. Protein 4.2 was not detected in guinea pigs (Cavia porcellus) (N = 14), Southern Brazilian swamp large rats (Myocastor coypus) (N = 2), cutias (Dasyprocta sp) (N = 4), and horses (Equus caballus) (N = 13). These animals also presented high ankyrin concentrations except for the horse which did not exhibit a sharp band, although minor components located between proteins 2 and 3 could account for the ankyrin family. The rodents studied did present band 6, which was not detectable in other common rodents such as white rats (Rattus norvegicus) (N = 9) and mice (Mus musculus) (N = 12). Since the absence of protein 4.2 does not disrupt the cytoskeleton membrane, we suggest that it is not an essential protein. Its absence may be compensated physiologically by the higher ankyrin concentration observed. Correspondence E.M. Guerra-Shinohara Av. Dr. Arnaldo, 355 01246-092 São Paulo, SP Brasil Fax: +55-11-853-3505 Presented at the XIII Annual Meeting of the Federação de Sociedades de Biologia Experimental, Caxambu, MG, Brasil, August 26-29, 1998. Research supported by CNPq. Publication supported by FAPESP. Part of a doctoral thesis presented by E.M. Guerra-Shinohara to the Departamento de Análises Clínicas e Toxicológicas, Faculdade de Ciências Farmacêuticas da Universidade de São Paulo. Received April 14, 1998 Accepted March 1, 1999

Human erythrocyte membrane proteins have been extensively studied, and band 3 and glycophorins have been classified as integral proteins, and the spectrins, band 2.1, band 4.1, band 4.2, band 4.5, band 4.9, band 5 (actin), band 6 (glyceraldehyde-3-phosphate dehydrogenase), and band 7 as peripheral proteins.The peripheral proteins which form the cytoskeleton are very important for maintaining the protein network just below the membrane lipid bilayer (1).In general, mammals present small variations in erythrocyte membrane protein concentration as well as in molecular weight (2).Variable spectrin and ankyrin concentrations have been reported in mice (3)(4)(5)(6)(7)(8), as well as a molecular weight variation of band 3 in non-human primates (9), its enrichment in llamas (10), and the absence of band 6 in rats and mice (11,12).
In the course of an extensive investigation of red cell membrane proteins (2), 44 selected representatives of mammalian species belonging to 13 orders from Brazilian Zoos and Brazilian Research Institutes were investigated (   mM sodium phosphate buffer, pH 8.0, containing the following protease inhibitors: 0.2 mM phenylmethylsulfonyl fluoride, 0.2 mM N-ethylmaleimide, 1.0 mM disodium ethylenediaminetetraacetate, 0.1 mM diisopropylfluorophosphate, 0.1 mM Na-p-Tosyl-Llysine chloromethyl ketone, 0.1 mM p-hydroxymercuribenzoic acid, and 1 µM pepstatin A. The ghosts were washed with the same buffer, solubilized by standard methods and submitted to SDS-polyacrylamide gel electrophoresis (SDS-PAGE), using 10% polyacrylamide gel (13) as well as an exponential gradient (3-17%, with piston) polyacrylamide gel (14).A ghost sample without protease inhibitors was also prepared.
Coomassie Blue (0.605 mM) in isopropanol:acetic acid:water (5:2:6) was used to stain the SDS gels.An Ultrascan XL laser densitometer (Pharmacia) was used with gelscan/92 software.Figure 1 illustrates the SDS-PAGE of the erythrocyte proteins from the animals which did not present protein 4.2 and shows that the patterns were constant in the presence of individual inhibitors and the pool of all inhibitors.
The red cell morphology of all animals lacking band 4.2 is shown in Figure 2. In comparison with human red cells, it can be seen that Equus caballus erythrocytes are microcytic and that Rodentia erythrocytes are similar to human except for those of Dasyprocta sp, which exhibit occasional stomatocytes.However, it is difficult to ascribe these morphological differences to the absence of protein 4.2.

®
Equus caballus horse of minor components located between proteins 2 and 3, which could account for an ankyrin family.Protein 4.2 has been considered to play an important role in cytoskeleton anchorage to the integral protein 3, together with ankyrin (band 2.1).Inaba and Maede (15) reported the finding of a putative membrane protein 4.2 between 4.1a and 4.1b of horse erythrocyte membrane.This, however, was not observed in our study.Bands 4.1a and 4.1b always remain together, and the hypothesis raised by these investigators of an undefined band between 4.1a and 4.1b may be due to an artifact since an antibody against 4.2 was not employed in their study.If a band 4.2 exists in horse erythrocytes, it should be detected as a protein of lower molecular weight than proteins 4.1a and 4.1b.
Protease inhibitors were employed in order to exclude the possibility that the absence of band 4.2 was due to proteolysis.However, since antibodies to band 4.2 were

Homo sapiens man
Myocastor coypus Southern Brazilian swamp large rat not used in the present study, the possibility of a band immunologically similar to the protein of band 4.2 but of different molecular weight cannot be ruled out.
Our data, however, do suggest that protein 4.2 is not an essential protein since its absence does not disrupt the cytoskeleton membrane.Physiologically its absence may be compensated for by the higher ankyrin concentration observed in the mammals lacking band 4.2.

Figure 2 -
Figure 2 -Red cell morphology of animals lacking protein 4.2.Human erythrocytes are used as a comparative sample.Magnification: 1000X.

Table 1 -
). Red cells were collected into acid-citrate-dextrose solution, washed in buffered saline and lysed in cold 5 Mammals whose erythrocyte membrane proteins were studied and their relative amount of protein 4.2.