Biomedical subjects

M K Pangburn

Publications and source records attributed to M K Pangburn.

At least 19 recordsLinked to original sources

PubMed1995

Tyrosine is a potential site for covalent attachment of activated complement component C3.

Activation of C3 results in the generation of metastable C3b, which has been shown to preferentially react with the hydroxyl groups of carbohydrates and with specific serine and threonine residues in proteins. In this study we have examined the reactivity of metastable C3b with the third type of hydroxyl group present in proteins, tyrosine (Tyr). The results demonstrated that Tyr reacts with the thioester of metastable C3b and that this reactivity was 11-fold better than that of threonine, 47-fold better than serine and 50-fold better than the reactivity of carbohydrates. Model peptides containing Tyr showed even higher reactivity than free Tyr, demonstrating that incorporation into peptide structures does not block C3b attachment. The site of attachment was found to be in the alpha'-chain of C3b and the bond was hydroxylamine sensitive, indicating an ester linkage. The stability of the C3b-Tyr complex was measured under physiological conditions (pH 7.4, 37 degrees C) and compared to the stability of other C3b complexes. C3b-Tyr decayed 50% in 19 hr at 37 degrees C, but C3b bound to a Tyr-containing peptide was more stable, exhibiting a t1/2 of 53 hr. The ester linked complexes C3b-IgG and C3b-glycerol were less stable each exhibiting a t1/2 of approximately 8 hr. As yet, only two specific C3b attachment sites on proteins have been identified, Ser1217 in C4b and Thr144 in IgG1. The present evidence demonstrates that Tyr residues are highly reactive and that the C3b-Tyr linkage is stable. The findings suggest that complexes involving tyrosine residues as the site of attachment will also be found.

Amino Acid Sequence↗

PubMed1994

Covalent attachment of human complement C3 to IgG. Identification of the amino acid residue involved in ester linkage formation.

C3 (native complement component 3) plays a central role in the activation of complement and in the transport and processing of immune complexes. Proteolytic activation of C3 exposes a highly reactive thioester bond which preferentially reacts with the hydroxyl groups of acceptor molecules on activators such as immune complexes or carbohydrates on microorganisms. Recently, a C3 attachment site has been localized on the CH1 domain of IgG1 between Val134 and Lys156. We have synthesized a series of peptide analogs of this region to identify the preferred residue for C3b (the proteolytically activated form of C3) attachment. The parent peptide included all 6 hydroxyl-containing amino acids present in the proposed binding site and was highly reactive with activated C3. The C3b-peptide complex was sensitive to hydroxylamine as was C3b-IgG indicating that both were ester-linked. The kinetic profile of hydrolysis of the C3b-peptide complex under physiologic conditions was found to be nearly identical to the profiles of C3b-IgG, C3b-IgG1, and C3b-glycerol complexes. Site-specific amino acid substitution of threonine and serine residues in the peptide indicated that, in contrast to the attachment site in C4b, little or no attachment occurred at serine residues. The threonine corresponding to Thr144 in the CH1 domain of IgG was found to be the major acceptor site for C3b. Thr148 was the second most reactive site on the peptide, but this residue is buried in native IgG.

Amino Acid Sequence↗

PubMed1994

Specificity of the thioester-containing reactive site of human C3 and its significance to complement activation.

The specificity of the thioester-containing site in three plasma proteins is regulated by elements of their protein structures other than the thioester bond itself. Human C4A and alpha 2-macroglobulin preferentially form amide linkages while human C3 primarily forms ester linkages with hydroxyl groups. We have examined the thioester in C3 and found evidence of strong preferences for certain carbohydrates, indications of selectivity for specific positions on those carbohydrates and a preference for terminal sugars in polysaccharides. A testable set of rules are derived from these findings which predict preferred attachment sites on polysaccharides. A computer model of the effect of different reactivities on activation of the alternative pathway of complement suggested that organisms might greatly alter their susceptibility to complement with small changes in carbohydrate structure. While a random selection of 20 biological particles showed no correlation between activation and C3b attachment efficiency, subsets of related organisms differing primarily in their surface polysaccharide exhibited stronger correlations. The strongest correlation occurred in a series of the yeasts (Cryptococcus neoformans) possessing capsular polysaccharides with one, two, three or four branching xylose sugars per repeating unit. These organisms exhibited capture efficiencies for metastable C3b from 12% (one-xylose strain) to 41% (four-xylose strain).

Alcohols↗

PubMed1994

Biologically active recombinant human complement factor H: synthesis and secretion by the baculovirus system.

A complete cDNA clone (4.3 kb) encoding human complement factor H (hCFH; 155 kDa) has been cloned into the pVL1393 baculovirus expression vector and transfected into Spodoptera frugiperda Sf9 insect cells. A biologically active (92 +/- 15%) 140-kDa protein was secreted into the medium with a yield of more than 5 mg/liter. This is the first report of synthesis of biologically active recombinant hCFH in a heterologous system.

Animals↗

PubMed1994

Regulation of alternative pathway complement activation by glycosaminoglycans: specificity of the polyanion binding site on factor H.

The discrimination between activators and nonactivators of the alternative pathway of complement depends on the affinity of the control factor H for C3b molecules covalently associated with the target. The affinity of factor H for the C3b-target complex is regulated by a positively charged site at or near the 13th short consensus repeat (SCR) domain of factor H. In this study we have analyzed the ability of different glycosaminoglycans and other negatively charged macromolecules to interact with the factor H polyanion recognition site and to enhance binding of H to the C3b-target complex. Strongest enhancement of factor H binding to zymosan-C3b was observed by the highly sulphated glycoconjugates dextran sulphate (m.w. = 5,000), heparin, chondroitin sulphate A and carrageenan (types III and IV). DNA also enhanced H binding. Removal of N-linked sulphates or reduction of the size of heparin decreased its enhancing effect on H binding. Little or no effect was seen with chondroitin sulphate C, keratan sulphate, hyaluronic acid, colominic acid (bacterial polysialic acid) or negatively charged polypeptides. The results show that the interaction of the polyanion binding site on factor H with glycosaminoglycans depends upon the number, orientation and polymeric arrangement of sulphate groups and suggest that most, but not all, sulphated glycosaminoglycans participate in the protection of host tissues from complement damage by promoting inactivation of tissue-bound C3b.

Anions↗

PubMed1992

Spontaneous thioester bond formation in alpha 2-macroglobulin, C3 and C4.

Purified alpha 2-macroglobulin and complement proteins C3 and C4 were treated with ammonia to break their intramolecular thioester bonds and reform the original free cysteinyl and glutamyl side chains. When this reaction was performed at low temperature a conformational intermediate was trapped which lacked a thioester, but which could refold to the native structure and spontaneously reform the thioester and full biological function. The findings suggest that these proteins may undergo spontaneous post-translational self-modification forming the thioesters without involvement of enzymes or high energy metabolites such as ATP.

Complement C3↗

PubMed1992

Substituted isocoumarins as inhibitors of complement serine proteases.

Inhibition of complement proteins D, B, C2, C1s, C1r, I, and the catalytic fragments Bb and C2a by substituted isocoumarins was investigated. 3,4-Dichloroisocoumarin, a general serine protease inhibitor, inhibited factor D, C1r, and C1s moderately with second-order inhibition constants (kobs/[I]) of 40 to 190 M-1 s-1, but it did not inhibit C2, factor B, C2a, or Bb. The best inhibitor for factors D and B was 4-chloro-7-guanidino-3-methoxyisocoumarin with kobs/[I] values of 250 and 290 M-1 s-1, respectively. Most isocoumarins did not inhibit C2 or C2a; only 4-chloro-3-isothiureidoalkoxyisocoumarins were slightly inhibitory. 3-Alkoxy-4-chloro-7-guanidinoisocoumarins inhibited C1r and C1s moderately. The best inhibitor for C1r and C1s was 4-chloro-3-(3-isothiureidopropoxy)isocoumarin with kobs/[I] values of 6,600 and 130,000 M-1 s-1, respectively. Fifty amino acid or peptide thioesters containing Arg or other amino acids at the P1 site were tested as substrates of factor I, however none was hydrolyzed. Isocoumarins substituted with chloro and basic groups such as guanidino and isothiureidoalkoxy inhibited factor I activity with its natural substrate C3b, but kobs/[I] values were low. 4-Chloro-3-ethoxy-7-guanidinoisocoumarin inhibited activation of the alternative pathway and, to a lesser extent, of the classical pathway in serum. Several other substituted isocoumarins also inhibited cobra venom factor-initiated activation of the alternative pathway in serum.

Amino Acid Sequence↗

PubMed1992

Spontaneous reformation of the intramolecular thioester in complement protein C3 and low temperature capture of a conformational intermediate capable of reformation.

Three human plasma proteins contain intramolecular thioester bonds: complement components C3 and C4 and alpha 2-macroglobulin. Their thioesters form when glutamine and cysteine residues react in the newly translated proteins and ammonia is released. We have reversed this reaction by treating C3 with ammonia to cleave the thioester and reform the original Gln and Cys. Thioester scission initiates a multistep conformational transition. One intermediate was sufficiently stable to be isolated by high performance liquid chromatography. It lacked native C3 functions and was shown to contain one free sulfhydryl group. Incubation of this ammonia-inactivated C3 intermediate in the absence of ammonia resulted in refolding to a native C3 conformation and recovery of thioester-dependent functions, as evidenced by: 1) return of hemolytic function, 2) return of autolytic cleavage of the alpha-chain, and 3) return of the ability to attach to surfaces during complement activation. Refolding and thioester reformation were dependent on a free SH group and were inhibited by HgCl2 and other thiol-specific reagents. Incubation of ammonia-inactivated C3 at 25 degrees C at pH 7.4 resulted in recovery of 70% of the original C3 function. Refolding and thioester reformation exhibited a Gibbs free energy of +5.2 kcal/mol and were favored over unfolding to the final inactive form. During reformation of native C3 from 14CH3NH2-treated C3, return of the native conformation was accompanied by release of radiolabel from the protein and return of hemolytic complement function. These results suggest that folding of C3 provides both the energy and environment necessary to react the Gln and Cys residues, release ammonia, and form the thioester bond.

Ammonia↗

PubMed1992

Prevention of C3 deposition by capsular polysaccharide is a virulence mechanism of type III group B streptococci.

Strains of type III group B streptococci isolated from patients with neonatal sepsis are generally resistant to complement-mediated phagocytic killing in the absence of specific antibody. It has been suggested that the resistance of type III group B streptococci to phagocytosis results from inhibition of alternative-complement-pathway activation by sialic acid residues of the type III polysaccharide. To better define the relationship between structural features of the type III capsule and resistance of type III group B streptococci to complement-mediated phagocytic killing, we measured deposition of human C3 on group B streptococcal strains with altered capsule phenotypes. C3 binding was quantified by incubating bacteria with purified human 125I-C3 in 10% serum. Wild-type group B Streptococcus sp. strain COH1 bound eightfold fewer C3 molecules than did either of two isogenic mutant strains, one expressing a sialic acid-deficient capsule and the other lacking capsule completely. Similar results were obtained when the incubation with 125I-C3 was performed in serum chelated with Mg-ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'- tetraacetic acid (MgEGTA), suggesting that the majority of C3 deposition occurred via the alternative pathway. In contrast to the wild-type strain, which was relatively resistant, both mutant strains were killed by human leukocytes in 10% serum with or without MgEGTA. We also measured C3 binding to 14 wild-type strains of type III group B streptococci expressing various amounts of capsule. Comparison of degree of encapsulation with C3 binding revealed a significant inverse correlation (r = -0.72; P less than 0.01). C3 fragments released by methylamine treatment of wild-type strain COH1 were predominantly in the form of C3bi, while those released from the acapsular mutant were predominantly C3b and those from the asialo mutant represented approximately equal amounts of C3b and C3bi. We conclude from these studies that the sialylated type III capsular polysaccharide inhibits alternative-pathway activation, prevents C3 deposition on group B streptococci, and protects the organisms from phagocytic killing.

Animals↗

PubMed1991

Localization of the heparin-binding site on complement factor H.

Factor H is a regulator of complement activation and, in this capacity, it prevents activation of the alternative pathway on host cells and tissues when it recognizes markers on these surfaces. This report describes the binding characteristics and location of the site on factor H that is responsible for host recognition. Factor H was found to bind a variety of polyanions, including heparin, heparan sulfate, dextran sulfate, and clusters of sialic acid. In heparin-agarose binding assays it exhibited an affinity for heparin only 2-fold weaker than that of antithrombin III. Factor H exhibited little or no affinity for polyaspartic acid or bacterial colominic acid (polysialic acid). Factor H (Mr 150,000 with approximate dimensions of 30 x 600 A) is composed of 20 highly homologous domains (SCRs) that are arranged as beads on a string. Polyanions were found to block a tryptic cleavage site in domain 15, and a photoaffinity-tagged heparin probe labeled the region between domains 12 and 15. Affinity chromatography of tryptic fragments on heparin-Sepharose confirmed that this region contained the heparin-binding site. CNBr cleavage at Met787 located between SCRs 13 and 14 split the photoaffinity-tagged region. Sequence analysis strongly suggests that domain 13 contains the primary site of polyanion binding. Factor H expresses its complement regulatory function through a site located in domains 4-6 where C3b binds. Thus, the polyanion-binding site that regulates the affinity of factor H for C3b appears to reside more than 200 A away from the C3b-binding site.

Amino Acid Sequence↗

PubMed1990

C3-cleaving membrane proteinase. A new complement regulatory protein of human melanoma cells.

Human melanoma cells resistant to killing by the R24 mAb and human complement rapidly degrade surface-deposited C3b (M. Panneerselvam, S. Welt, L. J. Old, C.-W. Vogel. 1986. J. Immunol. 136:2534). We report that C-resistant melanoma cells express a membrane proteinase that can cleave C3b, generating a cleavage product with a molecular mass of approximately 30 kDa. The C3-cleaving proteinase was identified on the melanoma cells by its cross-reaction with antiserum to p57, a C3-cleaving proteinase previously isolated from human E membranes (C. Charriaut-Marlangue, M. Barel, R. Frade. 1986. Biochem. Biophys. Res. Commun. 140:1113). Preincubation of the C-resistant melanoma cells with anti-p57 IgG or their F(ab')2 fragments increased their susceptibility to complement killing from 25% to approximately 50% and reduced the rate of C3b cleavage and the amount of the 30-kDa fragment generated on the cells. Anti-p57 IgG stained C-resistant melanoma cells by indirect immunofluorescence and precipitated a protein with an apparent molecular mass of 65 kDa. This membrane protein, termed p65, was not detectable on C-susceptible melanoma cells. Membrane extracts from C-resistant melanoma cells also showed C3-cleaving activity when incubated with purified C3 or C3b, similarly generating a C3 fragment of approximately 35 kDa. This fluid-phase C3 cleaving activity could be partially inhibited by anti-p57 IgG. These data suggest that p65 is a C3-cleaving proteinase, antigenically related to p57, that is expressed on C-resistant melanoma cells and responsible for the C resistance of these cells. We propose that the membrane-bound C3-cleaving proteinase represents another C regulatory protein protecting host cells against killing by C.

Blotting, Western↗

PubMed1990

Properdin binds to sulfatide [Gal(3-SO4)beta 1-1 Cer] and has a sequence homology with other proteins that bind sulfated glycoconjugates.

Properdin, which stabilizes the C3 convertase during the activation of the alternate complement pathway, contains amino acid sequence homologies with several proteins that bind sulfated glycoconjugates, including the adhesive protein thrombospondin and the leech salivary protein antistasin. This homology is based around the sequence Cys-Ser-Val-Thr-Cys-Gly-X-Gly-X-X-X-Arg-X-Arg. To determine if these homologous amino acid sequences are sulfated glycoconjugate-binding domains, purified native properdin, as well as activated properdin (a high molecular weight form of properdin), were examined for binding to various lipids in solid phase radioimmunoassays. Of the lipids tested, both native and activated properdin bind with high affinity only to sulfatide [Gal(3-SO4)beta 1-1 Cer], but not to comparable levels of cholesterol-3-SO4, or several neutral glycolipids, gangliosides, and phospholipids. Sulfatide binding by both forms of properdin is inhibited by dextran sulfate (Mr = 500,000) or fucoidan, whereas only the activated form is inhibited by dextran sulfate (Mr = 5,000) or heparin. Comparable levels of chondroitin sulfates A, B, and C, keratan sulfate, dextran (Mr = 90,000), or hyaluronic acid do not inhibit binding. Taken together, these data suggest that properdin, like antistasin and thrombospondin, binds sulfated glycoconjugates and supports the conclusion that the homologous sequences are sulfated glycoconjugate-binding domains.

Amino Acid Sequence↗

PubMed1990

A mechanism of activation of the alternative complement pathway by the classical pathway: protection of C3b from inactivation by covalent attachment to C4b.

In this work we studied the role of the classical pathway complement component C4b in the activation of the alternative pathway. It was found that nascent C3b attaches with high efficiency to C4b and that C3b in C4bC3b complexes is protected from inactivation by factors H and I. Activation of C3 by factors B and D in the presence of Mg2+ ions and excess C4b led to 35% incorporation of nascent C3b into C4bC3b complexes in the fluid phase. In comparison, when human IgG was tested as an acceptor under similar conditions, only 12% of generated C3b was incorporated into IgGC3b complexes. The half-life time of dissociation of C3b from purified C4bC3b complexes was approximately 2.3 h at 37 degrees C. C4b in these complexes protected C3b from inactivation as effectively as any known alternative pathway activator. Thus, C3b bound to C4b was tenfold more stable than free C3b or C3b bound to a nonactivating surface. In comparison, the protection provided by attachment to human IgG was only 67% of that of C4b. The results provide an explanation for observations of alternative pathway recruitment following classical pathway activation and for the stability of the classical pathway C5 convertase on surfaces which do not provide protection for C3b from factors H and I.

Complement C3-C5 Convertases↗

PubMed1990

Complement alternative pathway activation and control on membranes of human lymphoid B cell lines.

Membrane regulatory molecules normally prevent complement activation by autologous cells, therefore we compared the membrane control system of human lymphoid cell lines which activate or not human complement through the alternative pathway (AP). Membrane expression of decay-accelerating factor (DAF), membrane cofactor protein (MCP), complement receptors (CR)1, CR2 and H was measured either by radioimmunoassay or enzyme-linked immunosorbent assay on cell lysates. Soluble extracts of isolated membranes were tested functionally for their ability to accelerate the decay of C3bBb C3-convertase and allow the cleavage of C3b by factor I. Both regulatory functions were detected in solubilized membranes of Ramos cells, which do not activate the AP, as well as on the potent AP activator, Raji. Raji cells were found to express CR2, DAF and MCP molecules, while MCP was the only known regulatory protein detected on Ramos cells which expressed neither CR1, nor CR2, H or DAF. The I-cofactor activity of both Raji and Ramos cells was immunoprecipitated by anti-MCP, but the decay-accelerating activity was not adsorbed by anti-DAF nor by any of the available antibodies. Two EBV genome-negative cell lines (BJAB, BL41) were tested before and after in vitro conversion by EBV. As previously shown, EBV-converted cell lines activate the AP more efficiently than EBV- cell lines. At the same time, EBV superinfection induces an increase of both AP regulatory functions of cell membranes and enhances the expression of DAF, MCP and CR2. The results of this study show that complement activation by lymphoid cell lines is not related to an impaired autologous control of these cells, but that the expression of regulatory molecules increases together with the appearance of activating structures on the cell surface. Our results also suggest the occurrence of a new factor involved in the decay-accelerating activity on BL lines.

Antigens, CD↗

PubMed1990

Discrimination between activators and nonactivators of the alternative pathway of complement: regulation via a sialic acid/polyanion binding site on factor H.

The alternative complement pathway is capable of discriminating human cells and tissues from a wide variety of potential pathogens. It has been recently demonstrated that attachment of complement component C3b to activator-derived molecules (e.g., small polysaccharides) restricts inactivation of C3b by factors H and I in a manner similar to activator surfaces. It is now shown that restriction is reversed by certain soluble polyanions (e.g., sialoglycopeptides, heparin, or dextran sulfate) that mimic the effects of sialic acid and glycosaminoglycans on human cells and tissues. Fluid-phase polyanions enhanced binding of factor H to C3b attached to activating particles, indicating that the effect resulted from increased affinity between C3b and factor H. The enhancement was specific for activator-bound C3b since no enhancement was observed on nonactivating particles. While several polyanions could cause this effect, some polyanions could not, indicating specificity. The active polyanions also inhibited lysis of cells via the alternative pathway. The binding site for sialic acid appears to reside on factor H, since factor H bound to heparin-agarose and to sialic acid-bearing fetuinagarose, whereas C3b bound to neither under the same conditions. These observations suggest that occupation of a specific site on factor H by polyanions induces an increase in the C3b-H affinity, resulting in discrimination of host cells and tissues from alternative pathway-activating foreign cells.

Animals↗

PubMed1990

Reduced activity of DAF on complement enzymes bound to alternative pathway activators. Similarity with Factor H.

Attachment of C3b to activators of the alternative pathway of complement results in a decrease in regulatory activity expressed by Factor H. Decay-accelerating factor (DAF) and Factor H were found to exhibit quantitatively similar decreases in regulatory activity toward the C3 convertase (C3b,Bb) bound to activators, such as zymosan (Zym) and rabbit erythrocytes (ER), compared to non-activators, such as sheep (ES) and bovine (EB) erythrocytes. Purified DAF and Factor H, in 0.1% NP-40, were assayed by measuring the amount required to release 50% of the radiolabelled Bb in 10 min from C3b,Bb on Zym or cross-linked erythrocytes. The relative effectiveness (i.e. the restriction index, RI) of DAF for accelerating the decay of C3b,Bb on the various particles was: ES (1.0), ER (0.04) and Zym (0.03). The RI for Factor H was: ES (1.0), ER (0.04) and Zym (0.07). The rate of decay of C3b,Bb induced by DAF and Factor H showed similar restriction. The results suggest that the regulatory properties of DAF are reduced if the cells on which it resides become activators of the alternative pathway as a result of transformation, virus infection or surface alteration. These findings may explain reports of dysfunctional DAF on alternative pathway-activating cells.

Animals↗

PubMed1989

Analysis of the mechanism of recognition in the complement alternative pathway using C3b-bound low molecular weight polysaccharides.

The human complement (C) system recognizes bacterial, fungal and viral activators of the alternative pathway following covalent attachment of the protein C3b to carbohydrates (CHO) on the surface of the organisms. Recognition first manifests itself as a 3- to 10-fold reduction in the affinity of C3b for factor H, a regulatory protein of C. This report describes the use of a fluorimetric assay which is sensitive to the C3b-H interaction to study the characteristics of recognition. Fluid phase C3b covalently bound to CHO (C3b-CHO) was prepared by activating C3 in the presence of the small homopolymers dextran (alpha 1-6 polyglucose) or inulin (beta 1-2 polyfructose). In particulate form both polysaccharides are activators of C. The conjugates exhibited increased resistance to inactivation in the factor H-dependent assays compared to C3b not bound to CHO and to C3b bound to mono- or disaccharides. The dextran-induced restriction of inactivation was partially reversed by treatment of the conjugate with dextranase. C3b-CHO conjugates failed to bind to factor H-Sepharose and when introduced into serum behaved as though C3b was attached to particulate activators of C, suggesting that the fluorimetric assay accurately reports recognition. The results suggest that the recognition site which induces a reduction in the affinity of C3b for factor H is distinct from the thioester site of C3b and can recognize structural features of polysaccharides including size, sialic acid content, and possibly aspects of three-dimensional oligosaccharide structure.

Binding Sites↗

PubMed1989

Analysis of recognition in the alternative pathway of complement. Effect of polysaccharide size.

Covalent attachment of the complement (C) protein C3b to polysaccharides on biologic particles which activate the alternative pathway leads to changes in the affinity of C3b for factor H, a regulatory protein of the C system. In this study the size of the site with which the polysaccharides interact and its special relationship to the thioester site were investigated using a fluorimetric assay and soluble C3b attached to low m.w. polysaccharides. Oligomers of alpha 1-6 and alpha 1-4 polyglucose and beta 1-2 polyfructose were prepared and attached to C3b at the thioester site. C3b bound to monomeric, dimeric, or trimeric sugars exhibited the same interaction with factor H as free C3b, i.e., there was no effect due to attachment alone. Beginning with tetrameric oligosaccharides a linear decrease in factor H binding was observed with increasing oligosaccharide size and the effect reached an apparent maximum with large polysaccharides. Maximum inhibition of factor H function was estimated to occur at a length of 16 saccharide units. The results suggest that this site, which regulates the inactivation rate of surface-bound C3b and thus the activation of the alternative pathway of C, spans a maximum of 13 sugar units (less than 65 A) starting four units (approximately 15 A) from the thioester site in C3b.

Binding Sites↗