Biomedical subjects

A M Blom

Publications and source records attributed to A M Blom.

30 records · Page 2Linked to original sources

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↗

PubMed2000

Positively charged amino acids at the interface between alpha-chain CCP1 and CCP2 of C4BP are required for regulation of the classical C3-convertase.

C4b-binding protein (C4BP) is an abundant and potent down-regulator of complement activation. In its presence, the assembly of the classical pathway C3-convertase is prevented and its natural decay is accelerated. C4BP also acts as a cofactor to the serine proteinase factor I in the cleavage of C4b. C4BP contains repeats of small structural domains: complement control protein (CCP) modules. Previously, we constructed and purified nine recombinant C4BP molecules in which solvent exposed positively charged amino acids at the interface between CCP1 and CCP2 were mutated to polar glutamines. Several of these mutants showed lower binding ability for C4b. In the present investigation, the collection of mutants was tested with functional assays and we found a correlation between changes in the apparent affinity of C4BP mutants for C4b and their ability to down regulate the C3-convertase. Mutagenesis of R(39), K(63), R(64) and particularly H(67) resulted in impaired C4b binding paralleled by lost ability of the C4BP mutants to prevent C3-convertase assembly and to increase the decay rate of the C3-convertase. Furthermore, these amino acids were found to be crucial for the factor I cofactor activity of C4BP in fluid phase degradation of C4b. In conclusion, a cluster of positively charged amino acid residues at the CCP1-CCP2 interface is identified as functionally important in the regulation of the C3-convertase by C4BP.

Amino Acid Substitution↗

PubMed2000

Bikunin--not just a plasma proteinase inhibitor.

Bikunin is a plasma proteinase inhibitor that has received little attention in the past, probably because its activity towards various proteinases was found to be relatively weak in early work. It was recently discovered, however, that bikunin effectively inhibits a proteinase that seems to be involved in the metastasis of tumour cells--cell surface plasmin--and that a fragment of bikunin inhibits two proteinases of the coagulation pathway--factor Xa and kallikrein. Furthermore, it has been found that bikunin has other properties, such as the ability to modulate cell growth and to block cellular calcium uptake. Most of the bikunin in the blood occurs as a covalently linked subunit of the proteins pre- and inter-alpha-inhibitor. In this form bikunin lacks some of its known activities, and there is evidence that its release by partial proteolytic degradation may function as a regulatory mechanism. Although the physiological function of bikunin still remains to be established, current data suggest that this protein plays a role in inflammation. Further studies could therefore lead to results of therapeutical value.

Calcium↗

PubMed2000

A cluster of positively charged amino acids in the alpha-chain of C4b-binding protein (C4BP) is pivotal for the regulation of the complement system and the interaction with bacteria.

C4b-binding protein (C4BP) is a potent inhibitor of the classical pathway of complement. Structural analysis of our 3D model of the C4BP alpha-chain suggested that a cluster of positively charged amino acids at the interface between complement control protein (CCP) modules 1 and 2 could be involved in ligand binding. Nine C4BP mutants, where the positively charged amino acids were changed to glutamines, were expressed. We found that all of these displayed lower apparent affinity for C4b and that the site was also a specific heparin-binding site. The mutants demonstrated decreased ability to serve as factor I cofactors in a degradation of C4b. Also, their capacity to prevent the assembly of C3-convertase and to accelerate its decay were decreased. In conclusion, we found a binding site on C4BP that is functionally important for the regulation of C3-convertase. Many strains of Streptococcus pyogenes bind C4BP, via surface M proteins, which plays an important role in pathogenesis. Using the set of C4BP mutants we identified a key recognition surface for M proteins which overlaps with the C4b-binding site. The analysis of all mutants and inhibition with monoclonal antibodies indicated that the binding sites for C4b and M proteins are only overlapping but not identical. Furthermore, we found that the molecular mechanisms involved in these two interactions differ, since the binding between M proteins and C4BP is relatively insensitive to salt in contrast to the C4BP-C4b-binding. We suggest that binding between C4b and C4BP is governed mostly by electrostatic interactions, while additional non-covalent forces cause tight binding of C4BP to streptococcal M proteins.

Amino Acids↗

PubMed1999

A cluster of positively charged amino acids in the C4BP alpha-chain is crucial for C4b binding and factor I cofactor function.

C4b-binding protein (C4BP) is a regulator of the classical complement pathway, acting as a cofactor to factor I in the degradation of C4b. Computer modeling and structural analysis predicted a cluster of positively charged amino acids at the interface between complement control protein modules 1 and 2 of the C4BP alpha-chain to be involved in C4b binding. Three C4BP mutants, R39Q, R64Q/R66Q, and R39Q/R64Q/R66Q, were expressed and assayed for their ability to bind C4b and to function as factor I cofactors. The apparent affinities of R39Q, R64Q/R66Q, and R39Q/R64Q/R66Q for immobilized C4b were 15-, 50-, and 140-fold lower, respectively, than that of recombinant wild type C4BP. The C4b binding site demonstrated herein was also found to be a specific heparin binding site. In C4b degradation, the mutants demonstrated decreased ability to serve as factor I cofactors. In particular, the R39Q/R64Q/R66Q mutant was inefficient as cofactor for cleavage of the Arg937-Thr938 peptide bond in C4b. In contrast, the factor I mediated cleavage of Arg1317-Asn1318 bond was less affected by the C4BP mutations. In conclusion, we identify a cluster of amino acids that is part of a C4b binding site involved in the regulation of the complement system.

Amino Acids↗

PubMed1999

Structural characterization of inter-alpha-inhibitor. Evidence for an extended shape.

Inter-alpha-inhibitor (IalphaI) is a 180-kDa serum protein consisting of three polypeptides. Two of these, the heavy chains 1 and 2 (H1 and H2), are of 75-80 kDa and have similar amino acid sequences. The third polypeptide, bikunin, has a molecular mass of 25 kDa and contains a 7-kDa chondroitin sulfate chain that is covalently linked to the C-terminal amino acid residues of H1 and H2. IalphaI has been shown to be required for the formation of the hyaluronan-containing extracellular matrix of certain cell types. How IalphaI exerts this function is not known, but it appears that upon interaction with cells, the heavy chains are released and become covalently linked to hyaluronan. Our results indicate that IalphaI and its heavy chains are extended molecules; thus, upon electron microscopy, IalphaI appeared to consist of two globular domains connected by a thin structure 31-nm long and the isolated heavy chains of a globular domain and a "tail" about 15-nm long. Analysis of the heavy chains by partial proteolysis showed that the C-terminal halves are particularly sensitive to hydrolysis indicating that they are loosely folded. Furthermore, electron microscopy showed that partially degraded heavy chains lacked the extended regions. Taken together, these results suggest that the N-terminal half of the heavy chains forms a globular domain, whereas the other half has an extended and loosely folded structure.

Alpha-Globulins↗

PubMed1999

Binding of Zn(2+) to the plasma protein inter-alpha-inhibitor.

Inter-alpha-inhibitor (IalphaI) is a serum protein consisting of a chondroitin-sulfate-containing protein of 25 kDa (bikunin) and two other polypeptides of 75-80 kDa (heavy chains 1 and 2). The physiological function of IalphaI is unclear but recent results suggest that it is required for the formation of the extracellular matrix of certain cell types and that it has anti-inflammatory activity. It was previously reported that IalphaI isolated from serum contains bound Zn(2+), but details of this binding are lacking. Using equilibrium dialysis, we have found that when the free Zn(2+) concentration is raised from 0.3 to 50 micromol/L, the number of bound ions increases from 0.1 to 7. The concentration of free Zn(2+) in plasma is in the nanomolar range; our results therefore suggest that inter-alpha-inhibitor does not contain stoichiometric amounts of zinc ions under normal in vivo conditions.

Alpha-Globulins↗

PubMed1999

The complement regulator C4b-binding protein analyzed by molecular modeling, bioinformatics and computer-aided experimental design.

Molecular modeling and bioinformatics have gained recognition as scientific disciplines of importance in the field of biomedical research. Molecular modeling not only allows to predict the three-dimensional structure of a protein but also helps to define its function. Careful incorporation of the experimental findings in the structural/theoretical data provides means to understand molecular mechanisms for highly complex biological systems. C4b-binding protein (C4BP) is composed of one beta-chain and seven alpha-chains essentially built from three- and eight-complement control protein (CCP) modules, respectively, followed by a non-repeat carboxy-terminal region involved in polymerization of the chains. C4BP is involved in the regulation of the complement system and interacts with many molecules such as C4b, Arp, protein S and heparin. Here, we report experimental and computer data obtained for C4BP. Protein modeling together with site directed mutagenesis indicate that R39, R64 and R66 from the C4BP alpha-chain form a key binding site for heparin, suggesting that this region could be of major importance for interaction with C4b. We also propose that the first CCP of the C4BP beta-chain displays a key hydrophobic surface of major importance for the interaction with the coagulation cofactor protein S.

Complement C4b↗

PubMed1999

Structural prediction and analysis of endothelial cell protein C/activated protein C receptor.

The endothelial cell receptor (EPCR) for protein C (PC)/activated protein C (APC) is a 221 amino-acid residues long transmembrane glycoprotein with unclear physiological function. To facilitate future studies and to rationalize recently reported experimental data about this protein, we have constructed three-dimensional models of human, bovine and mouse EPCR using threading and comparative model building. EPCR is homologous to CD1/MHC class I molecules. It consists of two domains, which are similar to the alpha1 and alpha2 domains of MHC class I molecules, whereas the alpha3 domain of MHC is replaced in EPCR by a transmembrane region followed by a short cytosolic tail. The alpha1 and alpha2 domains of CD1/MHC proteins form a groove, which binds short peptides. These domains are composed of an eight-stranded antiparallel beta-pleated sheet with two long antiparallel alpha-helices. The distance between the helical segments dictates the width of the groove. The cleft in EPCR appears to be relatively narrow and it is lined with hydrophobic/aromatic and polar residues with a few charged amino acids. Analysis of the human EPCR model predicts that (a) the protein does not contain any calcium binding pockets; (b) C101 and C169 form a buried disulphide bridge, while C97 is free, and buried in the core of the molecule; and (c) four potential glycosylation sites are solvent exposed.

Amino Acid Sequence↗

PubMed1998

The C4b-binding protein-protein S interaction is hydrophobic in nature.

C4b-binding protein (C4BP) is a major regulatory molecule of the complement system. By forming a non covalent complex with the anticoagulant cofactor protein S (PS), it also plays an important role in blood coagulation. C4BP is composed of one beta-chain and seven alpha-chains that are essentially built from complement control protein (CCP)-modules. Our group has previously reported that the first (N-terminal) CCP module of the beta-chain (betaCCP1) contains the entire binding site for protein S. We now investigate further the binding of protein S to C4BP and show that the complex formation is essentially dependent on hydrophobic forces with minor contribution from electrostatic interactions. This result is in agreement with homology modeling experiments carried out in conjunction with inter-species sequence comparison and theoretical enumeration of potential binding sites. These methods pinpoint a solvent exposed hydrophobic cluster at the surface of the betaCCP1 module that is of crucial importance for the binding process.

Amino Acid Sequence↗

PubMed1997

Intracellular coupling of bikunin and the heavy chain of rat pre-alpha-inhibitor in COS-1 cells.

Pre-alpha-inhibitor is a serum protein consisting of two polypeptides: bikunin of 16 kDa, which carries an 8 kDa chondroitin sulphate chain, and heavy chain 3 (H3) of 74 kDa. The two polypeptides are linked through an ester bond between an internal N-acetylgalactosamine residue of the chondroitin sulphate chain and the C-terminal aspartic acid residue of H3. Both bikunin and H3 are synthesized by hepatocytes and become linked as they pass through the Golgi complex. H3 is synthesized with both N- and C-terminal extensions which are released during intracellular transport. To be able to analyse the assembly of pre-alpha-inhibitor in detail, we have cloned and sequenced the cDNA of rat H3. Upon expression of the protein in COS-1 cells, both propeptides were found to be released. Furthermore, co-expression of H3 and bikunin resulted in the two polypeptides becoming coupled, indicating that cells other than hepatocytes may have the capacity to form chondroitin sulphate-containing links.

Amino Acid Sequence↗

PubMed1997

Increase of bikunin and alpha1-microglobulin concentrations in urine of rats during pregnancy is due to decreased tubular reabsorption.

Bikunin and alpha1-microglobulin are two plasma proteins of about 25 kDa which are made in the liver from a common precursor. The concentration of bikunin in human urine has been shown to increase several fold during various conditions of stress. The mechanism behind this increase is unknown. We have studied pregnant rats and found that the bikunin and alpha1-microglobulin levels in their urine increased 3-fold towards the end of the pregnancy, whereas those of albumin and orosomucoid did not. There were no significant changes in either the bikunin/alpha1-microglobulin mRNA level or the concentrations of the two proteins in serum. These findings imply that the synthesis and the clearance rates of bikunin and alpha1-microglobulin are normal during pregnancy but that the tubular reabsorption of these proteins is decreased.

Alpha-Globulins↗