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Expression of carbohydrate residues in plasma membrane glycoproteins during the differentiation of amphibian epidermal cells

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Summary

Expression of various sugar residues on the plasma membrane of frog (Rana perezi) epidermal cells at different stages of differentiation has been monitored with the use of a battery of HRP-conjugated lectins. In paraffin-embedded tissue, mannose residues (stained by Concanavalin A) were detected at the keratinocyte cell surface in all epidermal strata. However,Lens culinaris agglutinin (LCA), also specific for mannose, specifically stained the plasma membrane of cells from the stratum germinativum. Expression of N-acetyl-glucosamine (GlcNAc), labelled with wheat germ agglutinin (WGA), was maximum at the cell surface of basal cells and progressively decreased through the stratum spinosum. Galactose (Gal) and N-acetyl-galactosamine (GalNAc) residues, labelled withGriffonia simplicifolia I (GS I) andGlycine max (SBA) agglutinins, respectively, were expressed according to the degree of differentiation in amphibian epidermal cells. Sialic acid-containing glycoproteins, labelled withLimax flavus agglutinin (LFA), were found in the outermost plasma membrane of the replacement cell layer and stratum corneum. Glycoproteins responsible for the observed lectin-binding patterns have been identified by staining on nitrocellulose filters after electrophoresis of solubilized plasma membrane fractions and Western blotting. Changes at the level of glycosylation of plasma membrane glycoproteins as epidermal cells differentiate are discussed on the basis of a progressive addition of Gal residues. Integral membrane proteins have been solubilized with the non-denaturing detergent CHAPS and glycoproteins containing terminal Gal residues, that are expressed according to the degree of differentiation in frog epidermis, have been partially purified by affinity chromatography on a GS I-Sepharose 4 B column. The purified fraction was composed by four acidic glycoproteins with isoelectric points between 4.6 and 5.2 and, in SDS-gels gave five major protein bands with approximate molecular weights of 148, 140, 102, 60, and 52 kDa in SDS-gels. The 102 and 52 kDa bands correspond to the a and β subunits of amphibian epidermal Na+,K+-ATPase as demonstrated by specific staining with a polyclonal antibody against the catalytic subunit of pig kidney proton pump and staining with lectins GS I, GS II, and WGA. Possible relationships between higher molecular weight proteins and the constituents of intramembranous particles from the outermost plasma membranes of the replacement cell layer and the stratum corneum are also discussed.

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Abbreviations

BSA:

bovine serum albumin

CHAPS:

(3-[(cholamidopropyl) dimethyl-ammonio] 1-propanesulfonate)

Con A:

Canavalia ensiformis agglutinin

DTT:

dithiothreitol

Gal:

galactose

GalNAc:

N-acetyl-D-galactosamine

GlcNAc:

N-acetyl-D-glucosamine

GS I:

Griffonia simplicifolia agglutinin I

GS II:

Griffonia simplicifolia agglutinin II

HRP:

horseradish peroxidase

LFA:

Limax flavus agglutinin

LCA:

Lens culinaris agglutinin

NDPAGIF:

non-denaturing polyacrylamide gel isoelectric focusing

PAGE:

polyacrylamide gel electrophoresis

PAP:

peroxidase-antiperoxidase

PBS:

phosphate buffered saline

PMSF:

phenyl methyl sulphonyl fluoride

RCL:

replacement cell layer

SBA:

soybean agglutinin (Glycine max)

SB:

stratum basal

SDS:

sodium dodecyl sulphate

SG:

stratum granulosum

SS:

stratum spinosum

UEA I:

Ulex europaeus agglutinin I

WGA:

wheat germ (Triticum vulgaris) agglutinin

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Authors and Affiliations

  1. Departamento de Bioquímica y Biologia Molecular, Universidad de Córdoba, Córdoba

    J. M. Roldán

  2. Departamento de Biología Celular, Facultad de Ciencias, Universidad de Córdoba, Avenida San Alberto Magno s/n, E-14004, Córdoba, Spain

    J. M. Villalba, F. Navarro, J. A. González-Reyes & P. Navas

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  1. J. M. Villalba
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  2. F. Navarro
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  3. J. M. Roldán
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  4. J. A. González-Reyes
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  5. P. Navas
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Villalba, J.M., Navarro, F., Roldán, J.M. et al. Expression of carbohydrate residues in plasma membrane glycoproteins during the differentiation of amphibian epidermal cells. Protoplasma 178, 87–96 (1994). https://doi.org/10.1007/BF01545959

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