Biomedical subjects

D A Cumming

Publications and source records attributed to D A Cumming.

At least 19 recordsLinked to original sources

PubMed1998

Solution structure and membrane interactions of the C2 domain of cytosolic phospholipase A2.

The amino-terminal, 138 amino acid C2 domain of cytosolic phospholipase A2 (cPLA2-C2) mediates an initial step in the production of lipid mediators of inflammation: the Ca2+-dependent translocation of the enzyme to intracellular membranes with subsequent liberation of arachidonic acid. The high resolution solution structure of this Ca2+-dependent, lipid-binding domain (CaLB) has been determined using heteronuclear three-dimensional NMR spectroscopy. Secondary structure analysis, derived from several sets of spectroscopic data, shows that the domain is composed of eight antiparallel beta-strands with six interconnecting loops that fits the "type II" topology for C2 domains. Using a total of 2370 distance and torsional restraints, the structure was found to be a beta-sandwich in the "Greek key" motif. The solution structure of cPLA2-C2 domain is very similar to the X-ray crystal structure of the C2 domain of phospholipase-C-delta and phylogenetic analysis clarifies the structural role of highly conserved residues. Calorimetric studies further demonstrate that cPLA2-C2 binds two Ca2+ with observed Kds of approximately 2 microM in an entropically assisted process. Moreover, regions on cPLA2-C2 interacting with membranes were identified by 15N-HSQC-spectroscopy of cPLA2-C2 in the presence of low molecular weight lipid micelles. An extended binding site was identified that binds the phosphocholine headgroup in a Ca2+-dependent manner and also interacts with proximal regions of the membrane surface. Based upon these results, a structural model is presented for the mechanism of association of cPLA2 with its membrane substrate.

Amino Acid Sequence↗

PubMed1998

Molecular cloning of human GDP-mannose 4,6-dehydratase and reconstitution of GDP-fucose biosynthesis in vitro.

We have cloned the cDNA encoding human GDP-mannose 4,6-dehydratase, the first enzyme in the pathway converting GDP-mannose to GDP-fucose. The message is expressed in all tissues and cell lines examined, and the cDNA complements Lec13, a Chinese Hamster Ovary cell line deficient in GDP-mannose 4,6-dehydratase activity. The human GDP-mannose 4,6-dehydratase polypeptide shares 61% identity with the enzyme from Escherichia coli, suggesting broad evolutionary conservation. Purified recombinant enzyme utilizes NADP+ as a cofactor and, like its E. coli counterpart, is inhibited by GDP-fucose, suggesting that this aspect of regulation is also conserved. We have isolated the product of the dehydratase reaction, GDP-4-keto-6-deoxymannose, and confirmed its structure by electrospray ionization-mass spectrometry and high field NMR. Using purified recombinant human GDP-mannose 4,6-dehydratase and FX protein (GDP-keto-6-deoxymannose 3,5-epimerase, 4-reductase), we show that the two proteins alone are sufficient to convert GDP-mannose to GDP-fucose in vitro. This unequivocally demonstrates that the epimerase and reductase activities are on a single polypeptide. Finally, we show that the two homologous enzymes from E. coli are sufficient to carry out the same enzymatic pathway in bacteria.

Amino Acid Sequence↗

PubMed1997

Solution structure of recombinant human interleukin-6.

Interleukin-6 (IL-6) is a 185 amino acid cytokine which exerts multiple biological effects in vivo and whose dysregulation underlies several disease processes. The solution structure of recombinant human interleukin-6 has now been determined using heteronuclear three and four-dimensional NMR spectroscopy. The structure of the molecule was determined using 3044 distance and torsion restraints derived by NMR spectroscopy to generate an ensemble of 32 structures using a combined distance geometry/simulated annealing protocol. The protein contains five alpha-helices interspersed with variable-length loops; four of these helices constitute a classical four-helix bundle with the fifth helix located in the CD loop. There were no distance violations greater than 0.3 A in any of the final 32 structures and the ensemble has an average-to-the-mean backbone root-mean-square deviation of 0.50 A for the core four-helix bundle. Although the amino-terminal 19 amino acids are disordered in solution, the remainder of the molecule has a well defined structure that shares many features displayed by other long-chain four-helix bundle cytokines. The high-resolution NMR structure of hIL-6 is used to rationalize available mutagenesis data in terms of a heteromeric receptor complex.

Antigens, CD↗

PubMed1996

Core2 beta-1,6-N-acetylglucosaminyltransferase enzyme activity is critical for P-selectin glycoprotein ligand-1 binding to P-selectin.

P-selectin glycoprotein ligand-1 (PSGL-1) is a high-affinity counterreceptor for P-selectin on myeloid cells and activated T-cells. In addition, PSGL-1 can serve, both in vitro and in vivo, as an E-selectin ligand. Appropriate glycosylation of PSGL-1 is crucial for binding to P-selectin. Functional PSGL-1 is known to bear sialyl lewis X (SLex) or a closely related oligosaccharide. In this study, we show that Chinese hamster ovary (CHO) cells expressing PSGL-1 and fucosyltransferase show a dramatic increase in binding to P-selectin when transfected with "core2" transferase, the enzyme that initiates branching of O-linked glycans. Moreover, only PSGL-1 from core2 transfectant CHO cells can be affinity-captured with P-selectin, suggesting that branched O-linked glycans are required for high-affinity binding to P-selectin. Analysis of PSGL-1-derived O-linked oligosaccharides produced in core2 transfected cells shows the presence of more elaborated glycans. Interestingly, transfection of core2 in these cells does not alter binding to E-selectin.

Animals↗

PubMed1996

Complete 1H, 15N and 13C assignments, secondary structure, and topology of recombinant human interleukin-6.

Essentially complete backbone and side-chain 1H, 15N and 13C resonance assignments for the 185-amino-acid cytokine interleukin-6 (IL-6) are presented. NMR experiments were performed on uniformly [15N]- and [15N,13C]-labeled recombinant human IL-6 (rIL-6) using a variety of heteronuclear NMR experiments. A combination of 13C-chemical shift, amide hydrogen-bond exchange, and 15N-edited NOESY data allowed for analysis of the secondary structure of IL-6. The observed secondary structure of IL-6 is composed of loop regions connecting five alpha-helices, four of which are consistent in their length and disposition with the four-helix bundle motif present in other related cytokines and previously postulated for IL-6. In addition, the topology of the overall fold was found to be consistent with a left-handed up-up-down-down four-helix bundle based on a number of long-range interhelical NOEs. The results presented here provide deeper insight into structure-function relationships among members of the four-helix bundle family of proteins.

Amino Acid Sequence↗

PubMed1996

A human colon carcinoma cell line exhibits adhesive interactions with P-selectin under fluid flow via a PSGL-1-independent mechanism.

It has been postulated that endothelial cell adhesion molecules involved in leukocyte recruitment play a role in metastasis. Using an in vitro flow model, we studied the adhesion of the human colon carcinoma cell line KM12-L4 to P-selectin, an inducible endothelial-expressed adhesion molecule involved in leukocyte recruitment. Recombinant forms of P-selectin and Chinese hamster ovary cells stably expressing P-selectin supported attachment and rolling of KM12-L4 cells at 1 to 2 dynes/cm2. The adhesive interactions to P-selectin were abolished by pretreatment of the KM12-L4 cells with neuraminidase but were unaltered by pretreatment of the KM12-L4 cells with O-sialoglycoprotein endopeptidase, an enzyme that cleaves mucin type glycoproteins such as P-selectin glycoprotein ligand-1 (PSGL-1). PSGL-1 is the only counter-receptor for P-selectin known to mediate myeloid cell adhesion to P-selectin under flow. Flow cytometric and Northern blot analyses revealed that KM12-L4 cells did not express PSGL-1 and monoclonal antibody PL1, a function-blocking monoclonal antibody to PSGL-1, had no inhibitory effect on KM12-L4 adhesion to P-selectin under flow. Compared with HL-60 cells, which express PSGL-1, the KM12-L4 cells exhibited a slightly lower rate of attachment to P-selectin and rolled at a significantly higher velocity. In summary, KM12-L4 human colon carcinoma cells interact with P-selectin, under flow, through a PSGL-1-independent adhesion pathway.

Animals↗

PubMed1995

A novel cobra venom metalloproteinase, mocarhagin, cleaves a 10-amino acid peptide from the mature N terminus of P-selectin glycoprotein ligand receptor, PSGL-1, and abolishes P-selectin binding.

Initial rolling of circulating neutrophils on a blood vessel wall prior to adhesion and transmigration to damaged tissue is dependent upon P-selectin expressed on endothelial cells and its specific neutrophil receptor, the P-selectin glycoprotein ligand-1 (PSGL-1). Pretreatment of neutrophils, HL60 cells, or a recombinant fucosylated soluble form of PSGL-1 (sPSGL-1.T7) with the cobra venom metalloproteinase, mocarhagin, completely abolished binding to purified P-selectin in a time-dependent and EDTA- and diisopropyl fluorophosphate-inhibitable manner consistent with mocarhagin selectively cleaving PSGL-1. A polyclonal antibody against the N-terminal peptide Gln-1-Glu-15 of mature PSGL-1 immunoprecipitated sPSGL-1.T7 but not sPSGL-1.T7 treated with mocarhagin, indicating that the mocarhagin cleavage site was near the N terminus. A single mocarhagin cleavage site between Tyr-10 and Asp-11 of mature PSGL-1 was determined by N-terminal sequencing of mocarhagin fragments of sPSGL-1.T7 and is within a highly negatively charged amino acid sequence 1-QATEYEYLDY decreases DFLPETEPPE, containing three tyrosine residues that are consensus sulfation sites. Consistent with a functional role of this region of PSGL-1 in binding P-selectin, an affinity-purified polyclonal antibody against residues Gln-1-Glu-15 of PSGL-1 strongly inhibited P-selectin binding to neutrophils, whereas an antibody against residues Asp-9-Arg-23 was noninhibitory. These combined data strongly suggest that the N-terminal anionic/sulfated tyrosine motif of PSGL-1 as well as downstream sialylated carbohydrate is essential for binding of P-selectin by neutrophils.

Amino Acid Sequence↗

PubMed1995

A sulfated peptide segment at the amino terminus of PSGL-1 is critical for P-selectin binding.

P-selectin glycoprotein ligand 1 (PSGL-1) is a mucin-like glycoprotein expressed on the surface of myeloid cells and serves as the high affinity counterreceptor for P-selectin. The PSGL-1-P-selectin interaction is calcium dependent and requires presentation of sialyl-Lewisx (sLex)-type structures on the O-linked glycans of PSGL-1. We report here the identification of a non-carbohydrate component of the binding determinant that is critical for high affinity binding to P-selectin. Located within the first 19 amino acids, this anionic polypeptide segment contains at least one sulfated tyrosine residue. We propose that this sulfotyrosine-containing segment of PSGL-1, in conjunction with sLex presented on O-linked glycans, constitutes the high affinity P-selectin-binding site.

Amino Acid Sequence↗

PubMed1995

P-selectin glycoprotein ligand-1 is the major counter-receptor for P-selectin on stimulated T cells and is widely distributed in non-functional form on many lymphocytic cells.

P-selectin glycoprotein ligand-1 (PSGL-1) is the high affinity counter-receptor for P-selectin on myeloid cells (Sako, D., Chang, X.J., Barone, K.M., Vachino, G., White, H.M., Shaw, G., Veldman, G.M., Bean, K.M., Ahern, T.J., Furie, B., Cumming, D. A., and Larsen, G. R. (1993) Cell 75, 1179-1186). Here we demonstrate that PSGL-1 is also widely distributed on T- and B-lymphocytic tumor cell lines, resting peripheral blood T and B cells, and on stimulated peripheral blood T cell and intestinal intraepithelial lymphocyte (IEL) lines. However, the majority of PSGL-1-positive resting peripheral blood lymphocytic cells and lymphoid tumor cell lines do not display significant P-selectin binding. In contrast, in vitro stimulated peripheral blood T cell and IEL lines avidly bind P-selectin, and PSGL-1 is the sole high affinity counter-receptor mediating this binding. During the course of in vitro stimulation, cell surface expression levels of PSGL-1 do not change as P-selectin binding increases. Rather, the activities of two glycosyltransferases reportedly involved in the production of functional PSGL-1 in myeloid cells are substantially higher in the stimulated T-lymphocytic lines than in resting T lymphocytes, consistent with the hypothesis that activation-dependent post-translational events contribute to the expression of functional PSGL-1 on lymphocytes.

Amino Acid Sequence↗

PubMed1995

Genomic organization and chromosomal localization of the gene encoding human P-selectin glycoprotein ligand.

The gene for P-selectin glycoprotein ligand (PSGL-1) has been cloned from a human placenta genomic DNA library. A single intron of approximately 9 kilobases was found in the 5'-untranslated region and the complete coding region resides in exon 2. The genomic clone differs from the cDNA clone isolated from HL-60 cells in that it encodes an extra copy of the decameric repeat located in the extracellular domain of PSGL-1. Further analysis indicated that the PSGL-1 genes of HL-60 and U-937 cells contain 15 repeats, whereas the PSGL-1 genes of polymorphonuclear leukocytes, monocytes, and several other cell lines contain 16 repeats. Transfection experiments did not indicate a functional difference between these two variants of PSGL-1. The two previously observed PSGL-1 mRNA species of 2.5 and 4 kilobases most likely arise from differential utilization of polyadenylation signal sequences. The organization of the PSGL-1 gene closely resembles those of CD43 and human platelet glycoprotein GPIb alpha, both of which have an intron in the 5'-noncoding region, a long second exon containing the complete coding region, and TATA-less promoters. The gene for human PSGL-1, which has been designated SELPLG by the Human Gene Nomenclature Committee, was mapped to chromosome 12q24 using Southern blot analysis of DNA from a set of human-mouse cell hybrids, and fluorescent in situ hybridization on metaphase chromosome spreads.

Amino Acid Sequence↗

PubMed1992

High level expression on a chimeric anti-ganglioside GD2 antibody: genomic kappa sequences improve expression in COS and CHO cells.

We report a flexible strategy for the high level expression of a recombinant human monoclonal antibody (mAb) in Chinese hamster ovary (CHO) cells, initially using COS monkey kidney cell transfections to evaluate rapidly modifications to immunoglobulin (Ig) DNA constructs. Using sequential transfections with two amplifiable markers, we generated CHO cell lines and clones that secrete 80-110 micrograms/10(6) cells/24 hours of a mouse-human chimeric IgG1 kappa mAb. This cellular productivity is considerably greater than most murine hybridomas and transfected myelomas. Our data also demonstrate that genomic kappa sequences can improve mAb expression in COS and CHO cells. As a paradigm, we focused our expression studies on a human chimeric form of 3F8, a murine mAb that binds to ganglioside GD2 on neuroblastoma and melanoma tumor cells.

Animals↗

PubMed1992

Physiological relevance of protein glycosylation.

The glycosylation of proteins is a complex biological pathway which is ordered and non-random. It is also a deterministic pathway dependent upon protein sequence, cellular phenotype, and the physiological environment. Two principal physiological roles have emerged within the past decade for protein-linked glycans: as recognition determinants and as modulators of various protein attributes such as bioactivity, pharmacokinetics, folding, and immunogenicity. All these attributes are crucial to development and application of protein-based pharmaceuticals. However, protein glycosylation represents a difficult structure/function problem since most glycoproteins exhibit microheterogeneity and oligosaccharides frequently contribute to this heterogeneity. Nevertheless, recent data suggest that different members of the heterogeneous ensemble exhibit distinguishable intrinsic properties, suggesting that the microheterogeneity of protein glycosylation represents a sophisticated mechanism of biological control.

Animals↗

PubMed1991

Glycosylation of recombinant protein therapeutics: control and functional implications.

The discovery, development, production and clinical application of recombinant glycoproteins for therapeutic administration in humans has been, and continues to be, an area of intensive scientific and medical effort. This effort has engendered considerable interest in the biological and therapeutic implications of post-translational modifications, particularly the most elaborated and sophisticated of these, protein glycosylation. As a result, numerous studies have appeared in the literature, especially within the past few years, which have greatly expanded our understanding of the biology of protein glycosylation. This review seeks to summarize these studies, illustrating that protein glycosylation, by modulating numerous biological attributes, is of central import in defining the utility of recombinant therapeutics.

Amino Acid Sequence↗

PubMed1991

Structural heterogeneity and functional differentiation: a rationale for glycosylation analysis of recombinant therapeutics.

Many secreted and membrane-bound proteins exhibit microheterogeneity, even after purification to 'homogeneity'. Posttranslational processing and modification, including glycosylation, often accounts for the observed heterogeneity. While microheterogeneity is traditionally viewed as a biochemical nuisance of little or no functional significance, the increasing weight of recent evidence suggests quite a different perspective: that structural heterogeneity, including microheterogeneity, can impart functional differentiation within the population and thus represents a sophisticated mechanism of biological control and functional diversification. Because of a presumed connection between functional attributes and clinical characteristics, this perspective is the fundamental rationale for the analysis of the glycosylation of proteins employed as therapeutic agents.

Amino Acid Sequence↗

PubMed1989

Separation of the complex asparagine-linked oligosaccharides of the glycoprotein fetuin and elucidation of three triantennary structures having sialic acids linked only to galactose residues.

Asparagine-linked oligosaccharides of the glycoprotein fetuin were isolated as reducing oligosaccharides after hydrazinolysis/re-N-acetylation/mild acid treatment of the Pronase-digested glycoprotein. The sialylated oligosaccharides were separated by high-performance liquid chromatography in two different systems, which resulted in greater than 35 fractions, comprising di-, tri-, tetra-, and pentasialylated oligosaccharides. The major components were isomeric structures comprising the tri- and tetrasialylated fractions. In this and the accompanying paper (Cumming et al., 1989), the structures of 10 of the major components of the tri-, tetra-, and pentasialylated oligosaccharide fractions are described. Separation protocols and three isolated structures having sialic acid linked only to galactose are presented in this paper.

Asparagine↗

PubMed1989

Structures of asparagine-linked oligosaccharides of the glycoprotein fetuin having sialic acid linked to N-acetylglucosamine.

In the accompanying paper (Bendiak et al., 1989), the separation of a series of oligosaccharides released from asparagine residues of fetuin was described. A series of NMR experiments, which included one- and two-dimensional nuclear Overhauser enhancement, two-dimensional correlation spectroscopy, and two-dimensional relayed-coherence spectroscopy, as well as permethylation analyses, established a Gal beta 1----3(NeuAc alpha 2----6)GlcNAc beta 1----4Man unit common to a series of purified structures. These oligosaccharides contained either three, four, or five glycosidically linked sialic acid residues. The NeuAc residue in alpha 2----6 linkage to GlcNAc gives rise to diagnostic chemical shift perturbations of particular proton signals in the oligosaccharides.

Acetylglucosamine↗

PubMed1989

Oligosaccharide-protein interactions: a three-dimensional view.

For carbohydrates to serve as recognition elements in cellular function, there must be 'receptors' which are capable of distinguishing between the multitude of oligosaccharide structures generated by a cell. Generally these receptors are assumed to be proteins, and the plant lectins have been used as model systems to examine the molecular basis for specificity in such interactions. Three aspects of the specificity of oligosaccharide-protein interactions will be discussed: (1) the conformational flexibility of oligosaccharides will be demonstrated through a quantitative analysis of nuclear magnetic resonance measurements; (2) a comparison of the measured and calculated values for the entropy barrier to oligosaccharide binding will be used to argue that the barrier arises from a loss of this conformational flexibility upon binding to the lectin (this conclusion is also supported by X-ray crystallographic studies); and (3) the thermodynamic model can be extended to the binding of glycoproteins to receptors and the high affinity of these interactions explained by either multivalency or fixation of the oligosaccharide in the 'correct' three-dimensional structure through interaction with the protein moiety.

Animals↗

PubMed1989

Separation of branched sialylated oligosaccharides using high-pH anion-exchange chromatography with pulsed amperometric detection.

Ten characterized sialylated oligosaccharides from bovine fetuin (B. Bendiak, M. Harris-Brandts, S. W. Michnick, J. P. Carver, and D. A. Cumming, Biochemistry, in press; and D. A. Cumming, C. G. Hellerqvist, M. Harris-Brandts, S. W. Michnick, J. P. Carver, and B. Bendiak, Biochemistry, in press) were chromatographed using high-performance anion-exchange chromatography with pulsed amperometric detection. At near neutral pH values, oligosaccharides were separated according to their number of formal negative charges from sialic acid; however, at alkaline pH, the neutral portion of the oligosaccharides enhanced resolution due to oxyanion formation. Specifically, trisialylated triantennary oligosaccharides containing a Gal-beta(1,3)GlcNAc sequence were more retained and could be completely separated from those having only Gal-beta(1,4)GlcNAc units. Oligosaccharides containing the same number of sialic acids were separated according to the combination of alpha(2,6)- and alpha(2,3)-linked sialic acids (alpha(2,6)-linked sialic acid reduced retention time). The relative molar electrochemical responses for di-, tri-, tetra-, and pentasialylated oligosaccharides were found to be similar (4.8 +/- 14% relative to glucose). Coelution studies were performed with each of the characterized oligosaccharides and the mixture of oligosaccharides which were released from fetuin with N-glycanase. The relative proportion of the major classes of sialylated oligosaccharides (bi-, tri-, tetra-, and penta-) varied significantly in bovine fetuin from different sources.

Chromatography, Ion Exchange↗