NOVEL ALPHA-GALNAC CONTAINING GLYCANS ON CYTOKERATINS ARE RECOGNIZED IN-VITRO BY GALECTINS WITH TYPE-II CARBOHYDRATE-RECOGNITION DOMAINS

We report on a novel posttranslational modification of cytoplasmic pro teins. Presented evidences suggest that cytokeratins are bound in vitr o by mammalian galectin-3 and the galectins from the sponge Geodia cyd onium via their type II carbohydrate recognition domains, whose highes t binding affinity is directed towards terminal alpha-N-acetylgalactos amine-bearing glycans with the general sequence GalNAc alpha 1-3Gal(NA c)beta. Specificity analyses and the characterization of the critical sugar residue on cytokeratins for galectin binding were done with cyto chemical and biochemical methods using various plant and animal lectin s. Binding of GalNAc-specific lectins was saturable, sensitive to mild periodate oxidation, inhibitable by glycoconjugates carrying terminal GalNAc, and abolished after treatment of the cytokeratins with alpha- N-acetylgalactosaminidase. Binding to bacterially expressed recombinan t cytokeratins did not exceed background binding. The presence of GalN Ac residues on highly purified cytokeratins from MCF-7 and HeLa SS6 ce lls was confirmed by sugar composition analyses using gas chromatograp hy/mass spectrometry. This novel posttranslational modification was no t restricted to cytokeratins of MCF-7 cells, but did also occur in all of 9 other examined human carcinoma cell lines and in a normal human mammary epithelial cell line. From these cytochemical and biochemical in vitro studies we hypothesize that this glycan with its terminal alp ha 1-3 linked GalNAc determinant might represent the first natural cyt oplasmic ligand for endogenous galectins-3 detected so far.