Purification of cranin, a laminin binding membrane protein. Identity with dystroglycan and reassessment of its carbohydrate moieties.
Cranin was described in 1987 as a membrane glycoprotein expressed in brain and many other tissues, which binds laminin with high affinity in a calcium-dependent manner. Dystrophin-associated glycoprotein ("dystroglycan") is a laminin-binding protein cloned in 1992 whose relation to cranin has remained uncertain. Here we describe the purification of cranin to homogeneity from sheep brain, show cranin to be a form of dystroglycan, and localize the N terminus of beta-dystroglycan to amino acid residue 654. We find that brain alpha-dystroglycan is tightly associated with membranes, and localizes to regions of synaptic contact as assessed by immunocytochemistry of rat cerebellum. Brain alpha-dystroglycan expresses high mannose/hybrid N-linked saccharides, terminal GalNAc residues, and the HNK-1 epitope. Although dystroglycan has previously been presumed to be a proteoglycan, the amino acid sequence, pI, O-sialoglycoprotease susceptibility, lectin-binding profile, and laminin-binding properties of brain dystroglycan are more typical of mucin-like proteins. Furthermore, using CHO mutant cell lines deficient in xylosyltransferase and galactosyltransferase I, which are required for glycosaminoglycan biosynthesis, it is shown that chondroitin sulfate and heparan sulfate are not critical for laminin binding, and indeed are apparently not expressed at all in dystroglycan from CHO cells.
Smalheiser
N R
NR
Department of Pediatrics, University of Chicago, Illinois 60637, USA.
Kim
E
E
D000595:Amino Acid Sequence; D000818:Animals; D001921:Brain / Q000378:metabolism*; D016466:CHO Cells; D002236:Carbohydrate Conformation; D002240:Carbohydrate Sequence; D002241:Carbohydrates / Q000032:analysis*; D002846:Chromatography, Affinity; D002848:Chromatography, DEAE-Cellulose; D006224:Cricetinae; D003598:Cytoskeletal Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D049030:Dystroglycans; D016189:Dystrophin / Q000378:metabolism; D006026:Glycoside Hydrolases; D015151:Immunoblotting; D007797:Laminin / Q000378:metabolism*; D037102:Lectins; D008562:Membrane Glycoproteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D008969:Molecular Sequence Data; D009439:Neuraminidase; D009844:Oligosaccharides / Q000737:chemistry* / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D012756:Sheep; D014162:Transfection
D002241:Carbohydrates; D003598:Cytoskeletal Proteins; D016189:Dystrophin; D007797:Laminin; D037102:Lectins; D008562:Membrane Glycoproteins; D009844:Oligosaccharides; D011994:Recombinant Proteins; D049030:Dystroglycans; D006026:Glycoside Hydrolases; D009439:Neuraminidase