Stimulation of B lymphocytes through their antigen receptor (BCR) results in rapid increases in tyrosine phosphorylation on a number of proteins and induces both an increase of phosphatidylinositol and mobilization of cytoplasmic free calcium. The BCR associates with two classes of tyrosine kinase: Src‐family kinase (Lyn, Fyn, Blk or Lck) and Syk kinase. To dissect the functional roles of these two types of kinase in BCR signaling, lyn‐negative and syk‐negative B cell lines were established. Syk‐deficient B cells abolished the tyrosine phosphorylation of phospholipase C‐gamma 2, resulting in the loss of both inositol 1,4,5‐trisphosphate (IP3) generation and calcium mobilization upon receptor stimulation. Crosslinking of BCR on Lyn‐deficient cells evoked a delayed and slow Ca2+ mobilization, despite the normal kinetics of IP3 turnover. These results demonstrate that Syk mediates IP3 generation, whereas Lyn regulates Ca2+ mobilization through a process independent of IP3 generation.

Muscle-eye-brain disease (MEB) is an autosomal recessive disorder characterized by congenital muscular dystrophy, ocular abnormalities, and lissencephaly. Mammalian O-mannosyl glycosylation is a rare type of protein modification that is observed in a limited number of glycoproteins of brain, nerve, and skeletal muscle. Here we isolated a human cDNA for protein O-mannose beta-1,2-N-acetylglucosaminyltransferase (POMGnT1), which participates in O-mannosyl glycan synthesis. We also identified six independent mutations of the POMGnT1 gene in six patients with MEB. Expression of most frequent mutation revealed a great loss of the enzymatic activity. These findings suggest that interference in O-mannosyl glycosylation is a new pathomechanism for muscular dystrophy as well as neuronal migration disorder.

SummarySignaling through the B cell antigen receptor (BCR) results in rapid increases in tyrosine phosphorylation on a number of proteins. The BCR associates with two classes of tyrosine kinase: Src-family kinase (Src-protein-tyrosine kinase [PTK]; Lyn, Fyn, Blk, or Lck) and Syk kinase. We have investigated the interaction between the Src-PTK and the Syk kinase in the BCR signaling. In contrast to wild-type B cells, BCR-mediated tyrosine phosphorylation of Syk and activation of its in vitro kinase activity were profoundly reduced in lyn-negative cells. The requirement of the Src-PTK to induce tyrosine phosphorylation and activation of Syk was also demonstrated by cotransfection ofsyk and src-PTK cDNAs into COS cells. These results suggest that the Src-PTK associated with BCR phosphorylates the tyrosine residue(s) of Syk upon receptor stimulation, enhancing the activity of Syk. The antigen receptor on B lymphocytes (BCR) is a surface immunoglobulin that associates with additional molecules involved in receptor transport and signal transduction (1-5). Stimulation of the BCR initiates a biochemical cascade in which protein-tyrosine kinase (PTK) activity is the earliest known event (6, 7). This PTK activation results in the tyrosine phosphorylation of several proteins, including the BCR Ig-ot, Ig-/3 chains (8), phosphatidylinositol (PI)-3 kinase (9, 10), guanosine triphosphate-activating protein (GAP) (11), protooncogene vav (12)(13)(14), and phospholipase c--r2 (15).Since the BCR complex does not have any intrinsic kinase activity, it is implicated that cytoplasmic PTK(s) is involved in initiating BCR signaling. One of the BCR-associated kinases is Src-PTK including Lyn, Fyn, Lck,. Recent evidence indicates that the activity of each of these enzymes is increased after cross-linking of the BCR (16). In addition to Src-PTK, BCR associates with the recently characterized Syk tyrosine kinase (19)(20)(21). Unlike the Src-PTK, Syk bears two SH2 domains and no NH2-terminal myristoylation site (20). Syk has been shown to be tyrosine phosphorylated and activated upon cross-linking of the BCR (21, 22). At present, it is not clear whether Syk activates the Src-PTK and/or vice versa, nor which of these two types of kinase plays a more important role through the BCR signaling. In this study, we focus upon how Src-PTK affects the activity of Syk through BCR signaling. Materials and MethodsCell Culture and DNA Transfection. COS-7 cells were cultured in DME containing 10% FCS. Wild-type and lyn-negative DT40 cells were cultured in RPMI 1640 supplemented with 10% FCS.Methods to establish lyn-negative DT40 cells were described in detail (23). Briefly, using gene targeting constructs including chicken genomic lyn and drug selection markers such as Neo, we created the lyn-negative cells by homologous recombination. Three alleles of lyn locus were sequentially disrupted and after isolating the three alleles targeted clone, we confirmed that this clone has incorporated a single copy of each construct. Transfection of lyn cDNA into the l...

A question of concentration: The condensation of ethyleneurea and formaldehyde can be controlled perfectly by the HCl concentration to provide either a hemicucurbit[6]uril 1, which functions as a host, or hemicucurbit[12]uril 2, which acts as a gelating agent, in yields of 94 and 93 %, respectively.

␣-Dystroglycan is a heavily glycosylated protein, which is localized on the Schwann cell membrane as well as the sarcolemma, and links the transmembrane protein ␤-dystroglycan to laminin in the extracellular matrix. We have shown previously that sialidase treatment, but not N-glycanase treatment, of bovine peripheral nerve ␣-dystroglycan greatly reduces its binding activity to laminin, suggesting that the sialic acid of O-glycosidically-linked oligosaccharides may be essential for this binding. In this report, we analyzed the structures of the sialylated O-linked oligosaccharides of bovine peripheral nerve ␣-dystroglycan by two methods. O-Glycosidically-linked oligosaccharides were liberated by alkaline-borotritide treatment or by mild hydrazinolysis followed by 2-aminobenzamide-derivatization. Acidic fractions obtained by anion exchange column chromatography that eluted at a position corresponding to monosialylated oligosaccharides were converted to neutral oligosaccharides by exhaustive sialidase digestion. The sialidases from Arthrobacter ureafaciens and from Newcastle disease virus resulted in the same degree of hydrolysis. The neutral oligosaccharide fraction, thus obtained, gave a major peak with a mobility of 3.8 -3.9 glucose units upon gel filtration, and its reducing terminus was identified as a mannose derivative. Based on the results of sequential exoglycosidase digestion, lectin column chromatography, and reversed-phase high-performance liquid chromatography, we concluded that the major sialylated O-glycosidically-linked oligosaccharide of the ␣-dystroglycan was a novel O-mannosyl-type oligosaccharide, the structure of which was Sia␣2-3Gal␤1-4GlcNAc␤1-2Man-Ser/Thr (where Sia is sialic acid). This oligosaccharide constituted at least 66% of the sialylated O-linked sugar chains. Furthermore, a laminin binding inhibition study suggested that the sialyl N-acetyllactosamine moiety of this sugar chain was involved in the interaction of the ␣-dystroglycan with laminin.