Biochemical and genetic analysis of an antigenic determinant found on N‐linked oligosaccharides in Dictyostelium

Abstract

Dictyostelium discoideum synthesizes many highly immunogenic carbohydrates of unknown structure and function. We have used monoclonal antibodies prepared against one of these called CA1 to investigate its structure and the consequences of its loss. CA1 is preferentially expressed on lysosomal enzymes as a specific arrangement of mannose-6-SO4 residues on N-linked oligosaccharides. Mutant strains HL241 and HL243 do not express CA1, and synthesize a truncated lipid-linked oligosaccharide (LLO) precursor that lacks the critical mannose residues needed for expression. The lesion appears to result from the loss of mannosyl transferase activity involved in LLO biosynthesis. The truncated LLO is poorly transferred to an artificial peptide acceptor in a cell-free N-glycosylation assay, and this appears to result from improper topological localization of the LLO or to a lower affinity of the LLO for the oligosaccharyl transferase. Although both mutants share these lesions, they are biochemically and genetically distinct. Only HL243 is lower in N-glycosylation in intact cells, and this is not a result of an altered structure of the LLO. There are other differences between the strains. HL241 can form fruiting bodies at a slower rate than normal while HL243 cannot aggregate. Genetic analysis of defects shows that the CA1 lesion in HL241 is recessive, while the lesion in both CA1 and in development are dominant and co-segregate in HL243 and are, therefore, likely to be in the same gene. Lysosomal enzyme targeting is normal but enzyme processing proceeds at a 2-3 fold slower rate in HL241 and HL243 compared to wild-type. Strain HL244 does not express CA1 since it completely lacks protein sulfation, but lysosomal enzyme targeting and processing proceeds at a normal rate, showing that sulfate is not essential for these processes. Alterations in oligosaccharide structure can have individualized effects on the biosynthesis of lysosomal enzymes. The results presented here illustrate how this approach can be used to study both the structure and function of carbohydrate epitopes.

Morphology and Dynamics of the Endocytic Pathway inDictyostelium discoideum

“…However, morphological descriptions of the membrane merger process occurring during endosome fusion are extremely rare. Study of cryo-immobilized and freeze-substituted D. discoideum cells at the EM level revealed that the membranes of some vacuoles were in tight contact and fusion events carbohydrate epitope present on D. discoideum lysosomal enzymes such as ␣-mannosidase and ␤-glucosidase (Knecht et al, 1984;Freeze et al, 1990), labeled small vesicular structures, that formed "rings of dots" around bigger, unlabeled vacuoles (marked by asterisks and also shown in higher magnification). Bar, 10 m. (C and D) Ultrastructure of late endosomes in wild-type D. discoideum cells that ingested BSA-Au particles for 60 min.…”

“…The following primary antibodies were used in this study: 1) a monoclonal antibody (mAb) 176-3-6 against coronin, an actin-binding protein (de Hostos et al, 1993) (gift from Dr. G. Gerisch, MPI for Biochemistry, Munich, Germany); 2) a mAb 221-342-5 (Neuhaus et al, 1998) against a common mannose-6-sulfate-containing carbohydrate epitope present on D. discoideum lysosomal enzymes such as ␣-mannosidase and ␤-glucosidase (Knecht et al, 1984;Freeze et al, 1990); 3) a mAb against horseradish peroxidase (HRP) from Vector Laboratories (Burlingame, CA); 4) a polyclonal antibody against cathepsin D (gift from Dr. J. Garin; CEA, Grenoble, France); 5) a mAb 221-1-1 against vacuolin (Jenne et al, 1998) (gift from Dr. M. Maniak, Abt. Zellbiologie, Universität GhK, Kassel, Germany); and 6) a mAb against a plasma membrane marker PM4C4 (mAb V4C4F3; Schwarz et al, 2000) (gift from Dr. J. Garin; CEA).…”

Molecular Mechanisms of Membrane Trafficking. What do we Learn from Dictyostelium discoideum?

“…The vacuolar-H+-ATPase ( ) is associated with membranes of the contractile vacuole complex and, to a much lesser extent with endosomes (monoclonal antibody 221-35-2 against the A-subunit of the vacuolar-H+-ATPase (Neuhaus et al 1998)). The common Antigen-1 ( • ) is a M-6-P-containing epitope shared by D. discoideum lysosomal enzymes such as (-mannosidase and (-glucosidase (Freeze et al 1990) (monoclonal antibody 221-342-5 (Neuhaus et al 1998)). Dynamin A (_) is a GTPase potentially involved in vesicle scission and is localised around post-lysosomal vacuoles in a punctate fashion (GFP-dynaminA (Wienke at al.…”

Evidence for a recycling role for Rab7 in regulating a late step in endocytosis and in retention of lysosomal enzymes in Dictyostelium discoideum.

“…Concerning the biosynthetic pathway, lysosomal enzymes are synthesized as precursor polypeptides (Mierendorf et al, 1985;Wood and Kaplan, 1985;Cardelli, 1986;Bush and Cardelli, 1989), and short N-terminal "fpro" regions, proteolytic processing, and the clathrin heavy chain are important in regulating targeting of hydrolases (Richardson et al, 1988;Ruscetti et al 1994;Ruscetti and Cardelli, unpublished results). N-linked mannose-rich oligosaccharide side chains and sulfate and probably phosphate sugar modifications only influence the rate of transport of the enzymes to lysosomes and not the fidelity of the targeting process Freeze et al, 1990).…”