Towards characterizing O-glycans: the relative merits of in vivo and in vitro approaches in seeking peptide motifs specifying O-glycosylation sites.
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Amino Acid Sequence↗
Biomedical subjects
K L Williams
Publications and source records attributed to K L Williams.
Characterization of O-linked glycosylation motifs in the glycopeptide domain of bovine kappa-casein.
kappa-Casein is the major glycoprotein in bovine milk. It has a proteinase-sensitive (chymosin) site which cleaves the glycoprotein into two segments: N-terminal para-kappa-casein domain and the C-terminal kappa-casein macroglycopeptide domain which is highly heterogeneous in oligosaccharide content. We have identified six sites of O-glycosylation on the macroglycopeptide by solid-phase Edman degradation: Thr121, Thr131, Thr133, Thr136 (A variant only), Thr142 and Thr165. No Ser residues are glycosylated. The glycosylation status of 15 of 17 potential O-glycosylation sites in the B variant was accurately predicted using the four peptide motifis previously proposed for the glycosylation of human glycophorin A (Pisano, A., Redmond, J.W., Williams, K.L. and Gooley, A.A., Glycobiology, 3, 429-435, 1993), provided one additional assumption is made concerning an inhibitory role for a nearby Ile.
Amino Acid Sequence↗
Optical flow analysis of the ventral cellular layer of the migrating Dictyostelium discoideum slug.
A digital image analysis system for extracting motion information from time-varying digital light microscopy images is presented. This system is then used to map out the movement profile of the surface layer of cells in contact with the substratum through the extracellular matrix (ECM) of the migrating Dictyostelium discoideum slug. From digital high magnification light microscopy images, the morphology of moving cells within the tail region of a young migrating wild-type WS380B slug is described, and compared with the morphology of streaming D. discoideum cells. It is shown that: (i) when the migrating tip of the slug touches the agar substrate, cells in the anterior ventral surface layer of the tip region slow dramatically; (ii) overall cell movement in the ventral surface layer of the migrating D. discoideum slug is slower than the movement of the slug as a whole; and (iii) in less than 10% of cases a wave of movement (groups of cells synchronously slowing down and then accelerating forward) propagates down the slug axis at approx. 1.2 microns s-1. The time interval between waves may be related to the time interval between tip-to-substratum contact that is periodically re-established during normal WS380B slug migration after each aerial projection of the tip.
Animals↗
A developmentally regulated glycoprotein complex from Dictyostelium discoideum.
The monoclonal antibody MUD50 recognizes an epitope on a family of developmentally regulated proteins in Dictyostelium discoideum (Alexander, S., Smith, E., Davis, L., Gooley, A., Por, S. B., Browne, L., and Williams, K. L. (1988) Differentiation 38, 82-90). This epitope is an O-linked oligosaccharide that requires the wild-type modB allele for expression. These glycoproteins are structurally diverse. Some are integral membrane proteins, while others are soluble and do not require detergent for solubilization. One of the soluble glycoproteins, PsB, is also recognized by the monoclonal antibody MUD102, which binds to the polypeptide backbone. The antibody recognizes a single 100-kDa band in Western analysis. In this report we demonstrate that the PsB molecule is part of a specific developmentally regulated multiprotein complex containing six different proteins. The complex is held together by both covalent and noncovalent bonds. Interestingly, only the PsB glycoprotein has the MUD50 O-linked oligosaccharide determinant. None of the proteins in the complex is N-glycosylated as assessed by sensitivity to peptide N-glycosidase F. Only one of the complex proteins is heavily phosphorylated. This work establishes the foundation for additional studies on the biosynthesis, processing, and assembly of the O-glycosylated PsB protein.
Animals↗
Post-translational modifications of the Dictyostelium discoideum glycoprotein PsA. Glycosylphosphatidylinositol membrane anchor and composition of O-linked oligosaccharides.
Prespore-specific antigen (PsA) is a cell-surface glycoprotein isolated from Dictyostelium discoideum, which is post-translationally modified by addition of carbohydrate to threonine residues of the carboxy-terminal peptide domain, and a glycosylphosphatidylinositol (GPI) anchor which attaches the glycoprotein to the cell membrane. The GPI anchor was isolated by proteolytic cleavage of the protein, and the structure of the lipid and glycan portions of the anchor were determined. The lipid moiety of the anchor is an inositolphosphoceramide which contains C18:0 phytosphingosine as a long chain base, and a mixture of fatty acids with a C18:1 mono-unsaturated fatty acid as the major component. The purified GPI anchor was susceptible to digestion by a bacterial phosphatidylinositol-specific phospholipase-C enzyme. The glycan of the GPI anchor consisted of two molecular species present in the ratio 55:45, the structures of which were determined by exoglycosidase sequencing and found to be Man alpha 1-2Man alpha 1-6Man alpha 1-4GlcNH2 and Man alpha 1-2Man alpha 1-2Man alpha 1-6Man alpha 1-4GlcNH2. The glucosamine in both structures is glycosidically linked to the inositol ring of the inositolphosphoceramide. The GPI glycan structures are consistent with the conserved core structure of all characterised GPI anchors, and the structure of the D. discoideum GPI moiety has features in common with structures from yeast, protozoa and higher eukaryotes. Compositional analysis of the carbohydrate attached to threonine residues in the carboxy-terminal peptide domain is also presented. The oligosaccharides bind to wheat germ agglutinin, and contain glucosamine and fucose as the major constituents.
Amino Acid Sequence↗
Pure populations of Dictyostelium discoideum prespore and prestalk cells obtained by flow cytometry have different redevelopment characteristics at their cell surfaces.
The multicellular slug stage of Dictyostelium discoideum consists of two major differentiated cell types: prespore and prestalk cells, which become, respectively, the spores and the stalk of the fruiting body. It is known that these cells, although expressing cell-type-specific proteins, remain totipotent, and experimental disruption of slugs results in redifferentiation taking place. We looked at what happens to cell-type-specific surface molecules when a cell changes from one type of another. Using monoclonal antibodies and flow cytometry we were able for the first time to obtain pure populations of single cells of each cell type. These were analysed during redevelopment. The initial hypothesis was that a proportion of each cell type would redifferentiate to reestablish the original proportions. However, it was found that the two cell types responded quite differently. Whereas almost all prestalk cells retained their prestalk surface antigen, in contrast, all prespore cells redifferentiated. During this process redifferentiating prespore cells simultaneously expressed surface determinants of both cell types, an event not seen in normal development.
Animals↗
Glycosylation sites identified by solid-phase Edman degradation: O-linked glycosylation motifs on human glycophorin A.
The human red blood cell sialoglycoprotein, glycophorin A (GpA), contains a 'mucin-like' extensively O-glycosylated extracellular domain which carries the MN blood group antigens. We have revised the sites of O-glycosylation in the extracellular domain of GpA by automated solid-phase Edman degradation, which allowed positive identification and quantitation of O-glycosylated Ser and Thr residues, as well as the single N-glycosylation site. One N-linked and 16 O-linked sites were identified. Carbohydrate was absent on Ser1, Ser14, Ser15, Ser23, Thr28 and Thr58 in GpA. We propose that the glycosyltransferases present in erythrocytes recognize specific flanking sequences around potential O-glycosylation sites. All 16 O-glycosylation sites are explained on the basis of four motifs. Three motifs are associated with Thr-glycosylation: Xaa-Pro-Xaa-Xaa where at least one Xaa = Thr; Thr-Xaa-Xaa-Xaa where at least one Xaa = Thr; Xaa-Xaa-Thr-Xaa where at least one X = Arg or Lys. The fourth motif is associated with Ser-glycosylation: Ser-Xaa-Xaa-Xaa where at least one Xaa = Ser. These simple rules explain the glycosylation (or lack of it) on 21 of 22 Ser/Thr in the extracellular domain of GpA.
Alleles↗
Patterns in Dictyostelium discoideum: the role of myosin II in the transition from the unicellular to the multicellular phase.
Dictyostelium discoideum amoebae which lack the myosin II gene are motile and aggregate to form rudimentary mounds, but do not undergo further morphological development (Manstein et al., 1989). Here we use scanning electron microscopy, light microscopy, immunofluorescence and computer analysis of time-lapse video films to study how D. discoideum myosin null cells of strains HS2205 and HS2206 aggregate. Myosin null cells are sufficiently coordinated in their movements to form two-dimensional aggregation streams, although mutant cells within streams lack the elongated shape and parallel orientation of wild-type strains. In the wild-type, cell movements are coordinated, cells usually joining streams that spiral inwards and upwards as the mound extends into the standing papilla. In the aggregates of mutant strains, cell movements are chaotic, only occasionally forming short-term spirals that rotate at less than half the speed of wild-type spirals and frequently change direction. Unlike the situation in the wild-type where spirals continue with mound elongation, cells within the mutant mound eventually cease translocation altogether as the terminal shape of the mound is reached and only intracellular particle movement is observed. Scanning electron micrographs show that the surface of the wild-type mound consists of flattened cells which fit neatly together. The myosin null cell mound has an uneven surface, the orientation of the cells is chaotic and no tip is formed. This is consistent with the results of synergy experiments in which myosin null cells were absent from the tips of chimeric HS2205/AX2 slugs and pre-culminates. Immunofluorescence microscopy using prespore and spore cell markers reveals that a prestalk/prespore pattern forms within the mutant mound but that terminal spore differentiation is incomplete. These results are discussed in relation to the role of myosin II in aggregation and morphogenesis.
Animals↗
Identification of a novel tyrosine kinase receptor-like molecule in neuroblastomas.
Growth factor receptors are important determinants of both normal and abnormal cell growth. We have now used degenerate primers designed from conserved tyrosine kinase domains to identify and clone a novel receptor-like molecule (designated Nbtk-1) from a NB41 mouse neuroblastoma cell line. Nbtk-1 is related to the met proto-oncogene family of tyrosine kinase receptors. Transcripts of approximately 2.1 and 2.6 kb have been found in mouse cell lines and one transcript of approximately 3 kb in human cell lines and in a wide range of primary human tumors, such as neuroblastomas, primitive neuroectodermal tumors (PNETs), Wilms' tumors, and melanomas and in the corresponding normal human tissues. These observations suggest that Nbtk-1 may have important roles in normal and tumor cell growth.
3T3 Cells↗
Length regulation in the Dictyostelium discoideum slug is a late event.
Time-lapse video light microscopy was used to study the emergence and maturation of the migratory slug from a D. discoideum aggregate. The anterior part, the tip of this simple multicellular organism, establishes migration prior to the definition of the rear, and hence the length of the slug. It was found that newly formed slugs of wild-type strain WS380B can reach lengths greater than 1 cm, yet mature slugs of this strain are rarely longer than 2-3 mumm. Often the tip extended out of the aggregation mound upon an arching pillar of cells. After the tip first touched the substratum, it commenced migration with a rapid succession of movement steps. Here we show that at the initiation of migration, a differential rate of cell movement along the developing slug axis results in a series of complicated changes, before the stable and mature shape of the slug is formed. Our results lead to new conclusions about D. discoideum slug formation and shape maintenance. Evidence is presented for regulation of slug length.
Animals↗
Putting the heat on sex determination.
Sex determination and differentiation are inherently fascinating to both layperson and geneticist. Major advances have accelerated interest in the molecular genetic events mediating these processes in nematodes, flies, mice and humans. Far less attention has been paid to those organisms, particularly reptiles, where sex is determined by environmental cues. However, recent experimental evidence suggests that the two modes of sex determination may not only share common genetic elements, but may also be regulated by similar mechanisms. We argue that the ability to manipulate sex by temperature provides a particularly suitable model for exploring the molecular basis of this fundamental biological process.
Animals↗
Dynamic arrays for fast, efficient, data manipulation during image analysis: a new software tool for exploratory data analysis.
Memory reallocation is used to construct a run-time data structure for fast/efficient storage of information during collection and analysis. The data structure presented uses dynamic memory but does not require the use of pointers to link nodes of information together. It allows for simple and efficient access to data via array indexing rather than through the use of lists or tree structures and it provides flexibility for competing storage requirements that are determined dynamically. The data structure is developed in the C programming language and a suite of ANSI standard C subroutines that make up a run-time data structure management system is provided.
Abstracting and Indexing↗
Size polymorphisms due to changes in the number of O-glycosylated tandem repeats in the Dictyostelium discoideum glycoprotein PsA.
The molecular weight polymorphism in the Dictyostelium discoideum cell surface glycoprotein PsA is due to incremental copies of an O-glycosylated tandem tetrapeptide repeat. Allelic variation at the pspA locus results in a PsA glycoprotein with three, four or five tandem copies of Pro-Thr-Val-Thr. The simplest explanation for the origin of this polymorphism is an unequal crossing over event in the ancestral gene containing four copies. Each Thr in the tandem repeat is substituted with carbohydrate, which is completely absent from PsA in strains carrying a glycosylation defective modB mutation. Glycosylated tandem repeats appear to be a common feature of cell surface glycoproteins which are characterized by short domains rich in Pro and Thr or Ser. It is probable that the glycosylated repeat domain acts as a "spacer" peptide that projects the globular domain above the glycocalyx.
Amino Acid Sequence↗
Tandem repeats in extrachromosomal ribosomal DNA of Dictyostelium discoideum, resulting from chromosomal mutations.
Extrachromosomal ribosomal DNA in the simple eukaryote Dictyostelium discoideum is readily separated from chromosomal DNA by orthogonal field electrophoresis (OFAGE), forming a prominent band in the 110-kb region of the gel. Here we show that mutations in at least two chromosomal genes give rise to a ladder of rDNA bands increasing in size up to about 300 kb. One of these mutations, the rrcA350 allele, which is recessive to wild type and maps to the centromere-proximal region of linkage group II, has an unstable phenotype; spontaneous revertants, which no longer exhibit the rDNA ladder, have been recovered. Another mutation rrc-351, provisionally mapped to linkage group IV, is dominant to wild type. The rDNA ladder is caused by concatamerization of a 34-kb fragment in the nontranscribed central spacer region of the 88-kb linear rDNA palindrome. Restriction enzyme analysis has revealed that each concatamer is generated by crossovers between two rDNA molecules.
Animals↗
Addressing sexual orientation in a public high school.
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Adolescent↗
Bleomycin resistance as a selectable marker for transformation of the eukaryote, Dictyostelium discoideum.
An expression cassette was constructed, which has the bacterial bleomycin (Bm) resistance-encoding gene (ble) fused to the Dictyostelium discoideum actin-6 promoter, with a segment of 3'-flanking DNA from the actin-8-encoding gene placed downstream from the ble gene to serve as a transcription terminator. Plasmid pMUW161, which contains this cassette and the D. discoideum plasmid Ddp2 origin of DNA replication, transformed D. discoideum with high efficiency under Bm selection. Hence, this construct is useful as a dominant selectable marker for D. discoideum.
Actins↗
Glycosylation sites identified by detection of glycosylated amino acids released from Edman degradation: the identification of Xaa-Pro-Xaa-Xaa as a motif for Thr-O-glycosylation.
Here we report the use of automated Edman degradation of covalently linked glycopeptides to identify positively the sites of O- and N-glycosylation. The O-glycosidic linkage of carbohydrate to the hydroxy amino acids Ser and Thr is a major form of post-translational modification. However, unlike Asn-linked glycosylation, which is identified by the consensus sequence Asn-Xaa-Thr/Ser, no simple motif conferring O-linkage to Thr and Ser has been described. After sequencing glycopeptides derived from two cell surface glycoproteins, a Thr-O-glycosylation motif of Xaa-Pro-Xaa-Xaa, where at least one Xaa = Thr(Sac), has been defined. This motif predicts the site(s) of Pro- associated Thr-O-glycosylation in O-glycosylated proteins, although it is clear that there are also other forms of Thr-O-glycosylation not associated with Pro.
Amino Acid Sequence↗
A comparison of the sensitivity and specificity of sialodacryoadenitis virus, Parker's rat coronavirus, and mouse hepatitis virus-infected cells as a source of antigen for the detection of antibody to rat coronaviruses.
Sialodacryoadenitis virus (SDAV) and Parker's rat coronavirus (PRC) are two recognized viral strains which cause spontaneous disease in the laboratory rat. Currently there is no recognized practical procedure which will accurately differentiate infections with these strains. Using SDAV- and PRC-infected L-2 cells as the source of antigen, and sera from rats collected post inoculation with either of these viral strains, the indirect fluorescent antibody (IFA) procedure was used to determine whether antibody titers could be used to differentiate infections from the homologous and heterologous virus. There was no detectable difference in the sensitivity or specificity of these systems in detecting antibody to the homologous or heterologous virus. Thus there was no evidence that SDAV- and PRC-infected cells would serve to differentiate antibody to the homologous virus using the IFA technique. In addition, antibody titers were similar when mouse hepatitis virus (MHV)-infected cells were used as the source of antigen for the IFA technique. However, using MHV or SDAV-infected cells as the source of antigen, there was a significant difference in antibody titers to the homologous virus detected using the immunoenzyme technique.
Animals↗