Christopher West, PhD - OUHSC Biochemistry & Molecular Biology

Skp1 is involved in many pathways of cellular regulation, including as an essential subunit of a protein complex which ubiqutinates cell cycle proteins and transcriptional factors for degradation. In the model organism Dictyostelium, we have found that Skp1 is modified by an unusual pentasaccharide attached to a hydroxyproline residue, leading to the discovery of an entirely novel glycosylation pathway in the cytoplasmic compartment of the cell. Skp1 glycosylation is correlated with its accumulation in the nucleus. Cloning of the glycosyltransferase genes using a proteomics approach is making possible detailed mechanistic studies to understand fundamental principles of glycosylation in the cytoplasm, and is providing insights into the evolution of the transferase genes. Recent studies indicate that prolyl hydroxylation of Skp1, a necessary prerequisite for glycosylation, is required for prestalk and prespore cell differentiation and an O2-dependent step in culmination. We are currently examining the hypothesis that conditional glycosylation of Skp1 is involved in quality control of Skp1 folding and entry into multi-subunit E3(SCF)ubiquitin ligases.

O-glycosylation of proteins usually occurs as they pass through the Golgi apparatus during their biosynthesis. In animals, mucin-type O-glycosylation, initiated via an a-linked GalNAc sugar attached to Thr- or Ser-residues, mediates many protein-specific functions. Unicellular eukaryotes, including pathogenic protozoans and Dictyostelium, form the potentially related GlcNAc-a-Thr/Ser linkage. Genes that encode the glycosyltransferases that modify Skp1 have led to the discovery of paralogs that mediate mucin-type O-glycosylation in the Golgi apparatus. This opens a new field of study to investigate the mechanism and function of O-glycosylation in microbial pathogens, such as trypanosomes, where mucin-type O-glycosylation is thought to be an important virulence factor. In addition, related predicted genes are found in pathogenic bacteria, suggesting that mucin-type O-glycosylation has a previously unsuspected ancient evolutionary heritage that originated in prokaryotes.

The spore coat of Dictyostelium is a model system for cellulose-basesd cyst walls of parasitic protozoans that are difficult to study experimentally. The Dictyostelium coat is an incredibly impervious protective layer assembled extracellularly at the cell surface from proteins, cellulose, and a Gal/GalNAc-containing polysaccharide. These components are organized as a polarized, 3-layered wall containing structural proteins sandwiched around a middle lamella of cellulose fibrils and the polysaccharide. They arrive via distinct pathways which raises important questions about how their delivery is synchronized and when they productively interact. Our studies are primarily focussed on the role of a trimeric complex of cellulose and two proteins, SP85 and SP65. SP85, a multidomain cellulose binding protein that is strategically positioned adjacent to the plasma membrane. Studies on mutant SP85s show that this protein influences timing of cellulose synthesis after protein secretion, organization of cellulose, and assembly of a structurally and functionally normal outer layer. Recent studies reveal that the polysaccharide is formed by novel two-domain Golgi glycosyltransferases and that the polysaccharide is essential for spore resistance to hypertonic stress. Future studies will examine the mechanisms by which SP85 and the polysaccharide contribute to spore coat assembly.

Links: For information on Dictyoselium discoideum, the model organism utilized in many of our studies, see www.Dictybase.org.

Photographs of Dictyostelium cells that have developed under normoxic or hypoxic conditions. Note that slugs culminate to form normal fruiting bodies in 21% O₂ but, at 10% O₂ , are inhibited from doing so. When subsequently transferred to 21% O₂ , the inhibited slugs complete development normally (not shown). Dictyostelium appears to use a mechanism of O₂ -sensing that is evolutionarily related to that of animals, which opens new avenues for the study of O₂ -regulation in both microorganisms and higher organisms.

van der Wel, H., A. Ercan, & C.M. West (2005) The Skp1 prolyl hydroxylase of Dictyostelium is related to the HIFa-class of animal prolyl 4-hydroxylases. J. Biol. Chem. 280:14645-14655.

Ercan, A. & C.M. West (2005) Kinetic analysis of a Golgi polypeptide-Thr N-acetyl-a-glucosaminyltransferase from Dictyostelium. Glycobiology 15:489-500.

West, C.M., H. van der Wel, P.M. Coutinho & B. Henrissat (2005) Glycosyltransferase genomics in Dictyostelium discoideum. In: Dictyostelium Genomics. Eds: W.F. Loomis & A. Kuspa (Horizon Scientific Press, Norfolk, UK) pp. 235-264.

Ketcham, C., F. Wang, S.Z. Fisher, A. Ercan, H. van der Wel, R.D. Locke, S. Doulah.k, K.L. Matta & C.M. West (2004) Specificity of a soluble UDP-galactose:fucoside a1,3galactosyltransferase that modifies the cytoplasmic glycoprotein Skp1 in Dictyostelium. J. Biol. Chem. 279:29050-29059.

He, C.Y., H.H. Ho, J. Malsam, C. Chalouni, C.M. West, E. Ullu, D. Toomre & G. Warren (2004) Golgi duplication in Trypanosoma brucei. J. Cell Biol. 165:313-321.

West, C. M., H. van der Wel, S. Sassi & E.A. Gaucher (2004) Cytoplasmic glycosylation of protein-hydroxyproline and its relationship to other glycosylation pathways. Biochim. Biophys. Acta 1673:29-44.

Xue, J., L. Song, S.D. Khaja, R.D. Locke, C.M. West, Roger A. Laine & K.L. Matta (2004) Determination of linkage position and anomeric configuration in Hex-Fuc disaccharides using electrospray ionization tandem mass spectrometry. Rapid Commun. Mass Spectrom. 18:1947-1955.

Wang, F., T. Metcalf, H. van der Wel & C.M. West (2003) Initiation of mucin-type O-glycosylation in Dictyostelium is homologous to the corresponding step in animals and is important for spore coat function. J. Biol. Chem. 278:51395-51407.

West CM, 2003: Evolutionary and functional implications of the complex glycosylation of Skp1, a cytoplasmic/nuclear glycoprotein associated with polyubiquitination. Cell Mol Life Sci. 60:229-240.

Metcalf T, Kelley K, Erdos GW, Kaplan L, West CM, 2003: Formation of the outer layer of the Dictyostelium spore coat depends on the inner-layer protein SP85/PsB. Microbiology. 149:305-317.

West CM, 2003: Comparative analysis of spore coat formation, structure, and function in Dictyostelium. Int Rev Cytol. 222:237-293.

West CM, Zhang P, McGlynn AC, Kaplan L, 2002: Outside-in signaling of cellulose synthesis by a spore coat protein in Dictyostelium. Eukaryot Cell. 1:281-292.

Van Der Wel H, Morris HR, Panico M, Paxton T, Dell A, Kaplan L, West CM, 2002: Molecular cloning and expression of a UDP-N-acetylglucosamine (GlcNAc):hydroxyproline polypeptide GlcNAc-transferase that modifies Skp1 in the cytoplasm of dictyostelium. J Biol Chem. 277:46328-46337.

Van Der Wel H, Fisher SZ, West CM, 2002. A bifunctional diglycosyltransferase forms the Fucalpha1,2Galbeta1,3-disaccharide on Skp1 in the cytoplasm of dictyostelium. J Biol Chem. 277:46527-46534.

West CM, van der Wel H, Gaucher EA, 2002: Complex glycosylation of Skp1 in Dictyostelium: implications for the modification of other eukaryotic cytoplasmic and nuclear proteins. Glycobiology. 12:17R-27R.

Van der Wel H, Morris HR, Panico M, Paxton T, North SJ, Dell A, Thomson JM, West CM, 2001: A non-Golgi a1,2-Fucosyltransferase that modifies Skp1 in the cytoplasm of Dictyostelium. J Biol Chem. 276:33952-33963.

Sassi S, Sweetinburgh M, Erogul J, Zhang P, Teng-umnuay P, West CM, 2001: Analysis of Skp1 glycosylation and nuclear enrichment in Dictyostelium. Glycobiology 11:283-295.

Zhang P, McGlynn AC, Loomis WF, Blanton RL, West CM, 2001: Spore coat formation and timely sporulation depend on cellulose in Dictyostelium.Differentation 67:72-79. [Journal issue cover photo]