CSM News Electronic Edition Volume 9, number 4 July 26, 1997 Please submit abstracts of your papers as soon as they have been accepted for publication by sending them to CSM-News@nwu.edu. Back issues of CSM-News, the CSM Reference database and other useful information is available at the Dictyostelium Web Page "http://dicty.cmb.nwu.edu/dicty/dicty.html" =================== Postdoc Available =================== POST-DOCTORAL RESEARCH ASSOCIATE POSITION. Molecular and cell biology of the contractile vacuole complex and water homeostasis in Dictyostelium discoideum. Two years guaranteed. Immediate start-up. Theodore L. Steck, M.D. Department of Biochemistry and Molecular Biology The University of Chicago 920 East 58th Street Chicago, IL 60637-1432 Phone: (773) 702-1329 Fax: (773) 702-0439 e-mail: t-steck@uchicago.edu =========== Abstracts =========== The Catalytic Subunit of Dictyostelium cAMP Dependent Protein Kinase: Role of the Amino Terminal Domain and of the Carboxy-terminal Residues in Catalytic Activity and Stability Lilian C. ETCHEBEHERE, Miguel X.P. van BEMMELEN, Christophe ANJARD, François TRAINCARD, Karine ASSEMAT, Christophe REYMOND* and Michel VÉRON Unité de Régulation Enzymatique des Activités Cellulaires, CNRS UMR 321, Institut Pasteur, 75724 Paris Cedex *Institut d'Histologie, Université de Lausanne , 9 Rue du Bugnon, CH-1005, Lausanne, Switzerland. Eur. J. Biochem., in press ABSTRACT The C subunit of Dictyostelium cAMP dependent protein kinase (PKA) is unusually large (73 kDa) due to the presence of 330 amino acids lying N-terminal to the conserved catalytic core. In contrast, the sequence following the core, including a C-terminal -Phe-Xaa-Xaa-Phe-COOH motif, is highly conserved. We have characterized the catalytic activity and stability of C subunits mutated in sequences lying outside the catalytic core and we have analyzed their ability to interact with the R subunit and with the peptide inhibitor PKI. Mutants carrying deletions at the amino-terminal domain displayed little difference in their kinetic properties and retained their capacity to be inhibited by R subunit and by PKI. In contrast, the mutation of one or both of the phenylalanine residues in the C-terminal motif resulted in a decrease of both catalytic activity and stability of the proteins. Inhibition by the R subunit or by PKI were however unaffected. Sequence comparison analysis of other protein kinases revealed that a -Phe-Xaa-Xaa-Phe- motif is present in many ser/thre protein kinases, although its location at the very end of the polypeptide is a particular feature of the PKA family. We propose that the presence of this motif may serve identify novel isoforms of protein kinases . --------------------------------------------------------------------- An IgG Monoclonal Antibody Against Dictyostelium discoideum Glycoproteins Specifically Recognizes Fuca1,6GlcNAcb in the Core of N-linked Glycans: Localized Expression of Core Fucosylated Glycoconjugates in Human Tissues Geetha Srikrishna, Nissi M. Varki, Peter C. Newell, Ajit Varki, and Hudson H. Freeze J. Biol. Chem., in press. Core fucosylation of N-linked oligosaccharides [GlcNAcb1,4(Fuca1,6)GlcNAcb1--Asn] is a common modification in animal glycans, but little is known about the distribution of core fucosylated glycoproteins in mammalian tissues. Two monoclonal antibodies, CAB2 and CAB4, previously raised against carbohydrate epitopes of Dictyostelium discoideum glycoproteins (Crandall, I.E. and Newell, P.C., Development 107, 87-94, 1989), specifically recognize fucose residues in a1,6 linkage to the asparagine-bound GlcNAc of N-linked oligosaccharides. These IgG3 antibodies do not cross-react with glycoproteins containing afucoses in other linkages commonly seen in N- or O- linked sugar chains. CAB4 recognizes core a1,6 fucose regardless of terminal sugars, branching pattern, sialic acid linkage or polylactosamine substitution. This contrasts to lentil and pea lectins that recognize a similar epitope in only a subset of these structures. Additional GlcNAc residues found in the core of N-glycans from dominant Chinese hamster ovary (CHO) cell mutants LEC14 and LEC18 progressively decrease binding. These antibodies show that many proteins in human tissues are core fucosylated, but their expression is localized to skin keratinocytes, vascular and visceral smooth muscle cells, epithelia and some extracellular matrix-like material surrounding subpopulations of lymphocytes. The availability of these antibodies now allows for an extended investigation of core fucose epitope expression in development and malignancy, and in genetically manipulated mice. --------------------------------------------------------------------- DICTYOSTELIUM LYSOSOMAL PROTEINS WITH DIFFERENT SUGAR MODIFICATIONS SORT TO FUNCTIONALLY DISTINCT COMPARTMENTS+ Glaucia M. Souza, Darshini P. Mehta, Marion Lammertz, Juan Rodriguez-Paris, Rongrong Wu, James A. Cardelli and Hudson H. Freeze J. Cell Sci., in press. Many Dictyostelium lysosomal enzymes contain mannose-6-phosphate (Man-6-P) in their N-linked oligosaccharide chains. We have now characterized a new group of lysosomal proteins that contain N-acetylglucosamine-1-phosphate (GlcNAc-1-P) linked to serine residues. GlcNAc-1-P containing proteins which include papain-like cysteine proteinases cofractionate with the lysosomal markers and are functional members of the endosomal/lysosomal pathway. Immunoblots probed with reagents specific for each carbohydrate modification indicate that the lysosomal proteins are modified either by Man-6-P or GlcNAc-1-P, but not by both modifications. Confocal microscopy shows that the two sets of proteins reside in physically and functionally distinct compartments. Vesicles with GlcNAc-1-P fuse with nascent bacteria-loaded phagosomes <3 min after ingestion, while those with Man-6-P do not participate in bacterial digestion until about 15 min after phagocytosis. Even though both types of vesicles fuse with phagosomes, GlcNAc-1-P and Man-6-P bearing proteins rarely colocalize. Since both lysosomal enzymes and their bound carbohydrate modifications are stable in lysosomes, a targeting or retrieval mechanism based on these carbohydrate modifications probably establishes and/or maintains segregation. --------------------------------------------------------------------- [End CSM News, volume 9, number 4]