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Biomedical subjects

J H Webb

Publications and source records attributed to J H Webb.

7 recordsLinked to original sources
PubMed2002

Studies on the interaction between vitamin K-dependent protein S and complement regulator C4b-binding protein: localization of binding sites and identification of a possible function of the complex.

Complement is a cascade-like system that is part of the innate immune defence. It is an explosive system, potentially harmful also for the host cells, and needs to be strictly regulated. An important down-regulator of complement is C4b-binding protein (C4BP). C4BP contains two different types of subunits, seven identical alpha-chains and one unique beta-chain. The alpha-chains bind to C4b, C4BP's target in the complement system. The beta-chain binds to vitamin K-dependent protein S. Approximately 70% of all protein S in plasma circulates in a high affinity complex with C4BP. Free protein S, the remaining 30%, functions as an important cofactor in the anticoagulant system. The reason for the complex formation between C4BP and protein S has remained an intriguing enigma. Protein S has a very high affinity to negatively charged phospholipids for protein S. One area where such phospholipids are present is the surface of the apoptotic cell, where the exposure of phosphatidylserine is an early event. Physiological apoptosis is characterized by a lack of inflammatory response in surrounding tissues, indicating that cells are rapidly cleared before leaking cytoplasmic components into the extracellular space. A number of studies demonstrate that early complement proteins are important for the removal of apoptotic cells, but that subsequent assembly of later complement components and anaphylatoxin release must be prohibited in order not to provoke an inflammatory response. We demonstrate that protein S localizes C4BP to the surface of apoptotic cells via binding to the exposed phosphatidylserine. The C4BP attached to the apoptotic cell through protein S was still able to bind C4b, suggesting that C4BP retains its physiological function also when localized to the apoptotic cell surface. In addition, we have also pinpointed a hydrophobic binding site for protein S on C4BP. The binding studies between C4BP and protein S were performed on recombinant proteins where mutations had been introduced. Mutations were chosen based on a 3D-homology model of the C4BP beta-chain.

Binding Sites↗
PubMed2001

Spawning success in Atlantic salmon (Salmo salar L.): a long-term DNA profiling-based study conducted in a natural stream.

Spawning success of Atlantic salmon (Salmo salar L.) was investigated, under near-natural conditions, in the Girnock Burn, an 8-km long tributary of the River Dee in Scotland. Employing minisatellite-based DNA profiling, mating outcomes were resolved over three spawning seasons by assigning parentage to progeny samples removed from spawning nests ('redds'). While individual spawning patterns differed markedly, consistent trends were present over the 3 years studied. Multiple spawning was found to be prevalent. More than 50% of anadromous spawners of both sexes contributed to more than one redd. Up to six redds for a single female and seven for a single male were detected. Both sexes ranged extensively. Distance between redds involving the same parent varied from a few metres to > 5 km. Distances > 1 km were common. Both males and females ranged to a similar extent. Range limit was not correlated to fish size. Pairs were not monogamous, both males and females mating with different partners at different sites. Size assortative mating was apparent among 1991 spawners but was not detected for 1992 or 1995. Redd superimposition was found to be common (17-22% of redds over the 3 years), although it was not correlated to the number of anadromous spawners present. High levels of nonanadromous mature parr mating success (40-50% of total progeny sampled) were recorded, and these likely contribute greatly to the effective population size. The relevance of these findings at the individual and population level is discussed, with particular reference to management implications.

Animals↗
PubMed2000

Localization of a hydrophobic binding site for anticoagulant protein S on the beta -chain of complement regulator C4b-binding protein.

C4b-binding protein (C4BP) is a plasma glycoprotein involved in regulation of the complement system. C4BP consists of seven alpha-chains and one unique beta-chain, all constructed of repeating complement control protein (CCP) modules. The beta-chain, made up of three CCPs, binds tightly to vitamin K-dependent protein S, a cofactor to anticoagulant activated protein C. When bound to C4BP, protein S loses its activated protein C cofactor function. In this study, we have mutated potentially important amino acids located at the surface of CCP1 of the beta-chain to probe the protein S-C4BP interaction. The substitutions were designed after analysis of a homology-based three-dimensional structure of the beta-chain and were L27T/F45Q, I16S/V18S, V31T/I33N, I16S/V18S/V31T/I33N, L38S/V39S, and K41E/K42E. The mutants were expressed in a prokaryotic system, purified using an N-terminal His-tag, refolded using an oxido-shuffling system, and tested in several assays for their ability to bind protein S. Our data define Ile(16), Val(18), Val(31), and Ile(33) as crucial for protein S binding, with secondary effects from Leu(38) and Val(39). In addition, Lys(41) and Lys(42) contribute slightly to the interaction. Our results further confirm that surface hydrophobicity analysis may be used to identify ligand recognition sites.

Base Sequence↗
PubMed2000

Human C4b-binding protein has overlapping, but not identical, binding sites for C4b and streptococcal M proteins.

Many strains of Streptococcus pyogenes bind C4b-binding protein (C4BP), an inhibitor of complement activation. The binding is mediated by surface M proteins in a fashion that has been suggested to mimic the binding of C4b. We have previously shown that a positively charged cluster at the interface between complement control protein domains 1 and 2 of C4BP alpha-chain is crucial for the C4b-C4BP interaction. To extend this observation, and to investigate the interaction with M proteins, we constructed and characterized a total of nine mutants of C4BP. We identified a key recognition surface for M proteins that overlaps with the C4b binding site because substitution of R64 and H67 by Gln dramatically reduces binding to both ligands. However, the analysis of all mutants indicates that the binding sites for C4b and M proteins are only overlapping, but not identical. Furthermore, M proteins were able to displace C4BP from immobilized C4b, whereas C4b only weakly affected binding of C4BP to immobilized M proteins. We found that the molecular mechanisms involved in these two interactions differ because the binding between M proteins and C4BP is relatively insensitive to salt in contrast to the C4BP-C4b binding. In addition, six mAbs directed against the alpha-chain interfered with C4b-C4BP interaction, whereas only two of them efficiently inhibited binding of C4BP to M proteins. Collectively, our results suggest that binding between C4b and C4BP is governed mostly by electrostatic interactions, while additional noncovalent forces cause tight binding of C4BP to streptococcal M proteins.

Amino Acid Substitution↗
PubMed1999

A lipid modified ubiquitin is packaged into particles of several enveloped viruses.

An anti-ubiquitin cross-reactive protein which migrates more slowly (6.5 kDa) by SDS-PAGE than ubiquitin was identified in African swine fever virus particles. This protein was extracted into the detergent phase in Triton X-114 phase separations, showing that it is hydrophobic, and was radiolabelled with both [3H]palmitic acid and [32P]orthophosphate. This indicates that the protein has a similar structure to the membrane associated phosphatidyl ubiquitin described in baculovirus particles. A similar molecule was found in vaccinia virus and herpes simplex virus particles, suggesting that it may be a component of uninfected cell membranes, which is incorporated into membrane layers in virions during morphogenesis.

African Swine Fever Virus↗