Confirmation by peptide sequence and co-expression on various cell types of the identity of CD44 and P85 glycoprotein

Abstract

The p85 glycoprotein expressed on a variety of human cell types including astrocytes and lymphocytes has not been associated with the CD44 cluster. The recent demonstration that Hermes, a glycoprotein implicated in the adhesion of lymphocytes to endothelium, belongs to the CD44 cluster raises interesting questions concerning the role of this molecule on astrocytes and on non-lymphoid cells. To obtain confirmation of the identity of p85 glycoprotein and CD44, p85 glycoprotein was purified from B-chronic lymphocytic leukemia cells by affinity to monolonal 50B4-IgG and electrophoretic elution, digested with trypsin or CNBr and fractionated by reversed-phase HPLC. The sequences of three peptides were obtained which could be aligned with the amino acid sequence deduced from the CD44 cDNA at residues 49-54, 59-66 and 309-323. These constitute the first reported peptide sequences for antigens of the CD44 cluster and confirm that p85 glycoprotein is indeed the product of the CD44 gene. Since two different cDNA clones encoding molecules with cytoplasmic tails of 72 and 5 amino acids have been isolated, the isolation of peptide 309-323 confirms the existence of a processed protein with the longer cytoplasmic domain. Using a cDNA probe, we have characterized the expression of CD44 in several normal and malignant cell types. The level of CD44 mRNA was correlated with the surface expression of CD44 antigens (50B4) in several leukemic cell lines, in astrocytoma lines and in normal granulocytes. Negative cells included the Y79 retinoblastoma line, the NALM-6 leukemic line and endothelial cells. Identical mRNA species of 5.0, 2.3 and 1.7 kb were present in all CD44-positive samples, including normal granulocytes, astrocytoma, melanoma and leukemia cell lines and leukemic cells from patients. The highest level of expression of CD44 was observed on astrocytoma lines and on acute lymphoblastic leukemia cells of immature phenotype. The presence of high levels of CD44 on malignant cells could increase the ability of these cells to adhere to matrix proteins and/or to interact with endothelium, thus potentially altering their capacity for invasiveness and metastasis.