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M K Pangburn

Publications and source records attributed to M K Pangburn.

At least 55 records · Page 3Linked to original sources
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↗
PubMed1989

Analysis of the natural polymeric forms of human properdin and their functions in complement activation.

Many of the anomalies observed in studies or properdin may be explained on the basis of its ability to form a series of multi-subunit polymers and by differences in the functions of these forms of properdin. Dimers (P2), trimers (P3), tetramers (P4), and higher Mr polymers (Pn) of the 46,000-Da subunit were separated by gel filtration or by cation exchange chromatography of purified properdin. The specific activity of each form was measured in two assays. The native properdin activity of P4 was 10 times that of P2 (on a molar basis) with the order: P4 greater than P3 greater than P2 greater than Pn. During C activation P4 was found to be consumed first, P3 second, and P2 last, consistent with their measured specific activities. Assays for activated properdin showed that only Pn caused fluid phase C consumption when incubated in serum at normal concentrations. Pn accumulated during long term storage of purified P and freezing rapidly converted the smaller oligomers to Pn. The isolated oligomers were extremely stable, but did redistribute after denaturation-renaturation cycles by using low pH or guanidine. Renaturation after exposure of any species to denaturing conditions yielded mixtures of 20:54:26 (P4:P3:P2). This distribution was almost identical to that found in fresh normal human serum or plasma, suggesting that a distinct distribution of oligomers exists in blood that provides the C system with an apparently advantageous range of specific activities.

Chromatography, Gel↗
PubMed1987

A fluorimetric assay for native C3. The hemolytically active form of the third component of human complement.

The content of native C3 in samples of purified C3 may be accurately determined using the fluorescent probe ANS (8-anilino-1-napthalene sulfonate). The assay is based on the 11.5-fold increase in fluorescence intensity of ANS which accompanies proteolytic conversion of native C3 to C3b. The assay may be performed in the presence of hemolytically inactive derivatives of C3 such as C3b and C3(H2O). It exhibits the unique feature of being independent of protein concentration and it does not require a C3 standard, other purified complement components, C3 depleted serum, cells or cell-bound intermediate complexes, such as EAC142. A method utilizing cation exchange chromatography (Mono S, Pharmacia) is also described for the rapid (30 min) analytical or preparative separation of native C3 from inactive forms of C3 and from C3 fragments.

Chromatography, Ion Exchange↗
PubMed1987

Specific deposition of complement protein C3b on abnormal PNH erythrocytes permits their separation by partitioning. Possible general approach for isolation of specific cell populations.

The deposition of complement proteins on a cell surface has previously been shown to reduce the cell's partition ratio in a two-polymer aqueous phase system. This phenomenon has now been extended to segregate, by partitioning, subpopulations of erythrocytes from patients with paroxysmal nocturnal hemoglobinuria (PNH). Purified components of the complement system were employed to deposit the protein C3b specifically on abnormal erythrocytes which lacked the membrane-associated complement regulatory protein DAF. As few as 2100 C3b/cell reduced the partition ratio and 24,000 C3b/cell resulted in resolution of the C3b-bearing and non-bearing human red cells. It was found that the proportion of cells separated did not equal the proportion of cells lysed by complement in the acidified serum lysis test when blood from three of the five patients was examined. The results indicate that the defect giving rise to DAF- cells may be, but is not necessarily, coexpressed with defects affecting other membrane-associated regulatory factors. A broader application of the method using monoclonal antibodies to direct purified complement components to specific cell populations should permit their isolation in large quantities.

CD55 Antigens↗
PubMed1986

Biosynthesis and glycosylation of the human complement regulatory protein decay-accelerating factor.

The biosynthesis and oligosaccharide structure of the human complement regulatory glycoprotein decay-accelerating factor (DAF) were studied in erythrocytes and cell lines. Initial information relative to carbohydrate moieties of DAF was obtained by enzymatic digestions. The 74,000 Mr erythrocyte DAF was lowered 3000 by endoglycosidase F, whereas endoglycosidase H had no effect, indicating one N-linked complex-type unit. Treatment with endo-alpha-N-acetylgalactosaminidase to remove O-linked oligosaccharides resulted in a 48,000 Mr molecule (67% of the Mr shift being due to sialic acid), which decreased to 45,000 Mr after sequential endoglycosidase F treatment. To additionally define the oligosaccharide structure and identify precursors in biosynthetic pathways, DAF was studied in the HL-60 cell line differentiated by vitamin D toward monocytes. Pulse-chase experiments with [35S]methionine revealed a precursor species of 43,000 Mr that underwent an early post-translational modification to a 46,000 Mr intermediate, and subsequently was chased into a mature species of 80,000 Mr that aligned with 125I surface-labeled DAF from these cells. All three forms of DAF were approximately 3000 lower in Mr in the presence of tunicamycin. The two lower Mr DAF species were sensitive to endoglycosidases F and H but not to neuraminidase or endo-alpha-N-acetylgalactosaminidase. In summary, DAF is synthesized as a 43,000 Mr precursor species containing one N-linked high-mannose unit. Before entering the central region of the Golgi, it is converted to a 46,000 Mr species by an as yet unknown post-translational modification. The 46,000 Mr form is converted to the 74,000 Mr (erythrocyte) or 80,000 Mr (leukocyte) membrane form of DAF by the addition of multiple, sialylated O-linked oligosaccharide chains (responsible for the large electrophoretic mobility shift) and conversion of the single N-linked high-mannose unit to a complex-type structure. The cell-specific Mr variation between red and white blood cells arises during this post-translational modification from the 46,000 Mr biosynthetic intermediate to the mature DAF species expressed on the cell surface.

Blood Proteins↗
PubMed1986

The C3 convertase of the alternative pathway of human complement. Enzymic properties of the bimolecular proteinase.

The association of Factor B with C3b (the major fragment of complement component C3) in the presence of Mg2+ results in the formation of a bimolecular zymogen, C3b,B, which is activated by the serine proteinase Factor D, generating the C3 convertase, C3b,Bb (EC 3.4.21.47). Cleavage of native C3 by the C3 convertase was monitored by recording the increase in fluorescence associated with C3b formation in the presence of the fluorescent probe 8-anilinonaphthalene-1-sulphonate. Measurements of initial rates of C3b formation at various C3 concentrations were analysed in accordance with the Michaelis-Menten equation, yielding kcat. = 1.78 +/- 0.08 s-1, Km = 5.86 X 10(-6) M and turnover number = 107 min-1. The assay was used to measure the Ki values of a variety of proteinase inhibitors. The C3 convertase has a short half-life, owing to spontaneous dissociation of the complex. The t1/2 and kcat./Km of the enzyme were determined by fitting an equation modelling both the kinetic reaction and enzyme decay to the fluorimetrically measured progress curve. The enzyme, C3b,Bb, exhibited a t1/2 of 90 +/- 2 s and a kcat./Km of 31.1 X 10(4) +/- 0.8 X 10(4) M-1 X s-1 at physiological pH, ionic strength and temperature. The enzyme that initiates activation of the alternative pathway, C3(H2O),Bb, was also examined. It was slightly less stable (t1/2 = 77 +/- 3 s) and exhibited only half the activity of C3b,Bb (kcat./Km = 16.3 X 10(4) +/- 1.0 X 10(4) M-1 X s-1).

Complement Activating Enzymes↗
PubMed1986

Differences between the binding sites of the complement regulatory proteins DAF, CR1, and factor H on C3 convertases.

Binding studies using purified decay-accelerating factor (DAF), CR1, and Factor H indicate that the primary interaction of DAF with C3 convertases is with the Bb or C2a subunits, whereas CR1 and Factor H interact primarily with the C3b or C4b subunits. The ability of soluble DAF, CR1, or Factor H to decay C3b,Bb bound to zymosan was inhibited by various concentrations of fluid-phase competitors (C3b, Bb, C3b,Bb, C3b,B, C4b, or C4b,C2a) in 0.1% NP-40 at 22 degrees C. The apparent association constants (appKa) for DAF were 0.045, 0.067, 0.91, 0.71, 0.00045, and 0.53 microM-1, respectively. The appKa for CR1 were 0.50, 0.0040, 1, 1, 1, and 1.1 microM-1, respectively. The appKa for Factor H were 4.3, 0.0005, 2.9, 6.3, 0.27, and 0.29 microM-1, respectively. Thus, C3b binds to DAF with a 10-fold lower affinity than to CR1 and a 100-fold lower affinity than to Factor H. The appKa of C3b,Bb for the three proteins were more similar: DAF (0.91 microM-1), CR1 (1 microM-1), and Factor H (2.9 microM-1). DAF binds to Bb with a 50% higher affinity than to C3b, and to C4b,C2a with a 1000-fold higher affinity than to C4b alone. In contrast, CR1 and Factor H bind almost equally well to the C3 convertases and to their noncatalytic subunits. The affinity of DAF for CVF,Bb was similar to its affinity for Bb alone, suggesting that DAF does not recognize conformational determinants unique to Bb in C3 convertases.

Binding Sites↗
PubMed1985

Detection of a neoantigen on human C3bi and C3d by monoclonal antibody.

A neoantigen was detected on human C3bi and C3d by using the monoclonal antibody (MoAb) 130. The antibody bound to EC3bi and EC3d cells but not to EC3b. Although highly purified C3bi or C3d strongly inhibited the binding of the antibody to EC3d, highly purified C3c had no such effect. Native C3, C3b, or C3(H2O) inhibited this binding only weakly. The neoantigen was also detected in serum after activation with zymosan or heat-aggregated IgG, and it was found bound to the aggregated IgG and zymosan particles. Plasma samples from patients with immunologic disorders were tested for this neoantigen, and 25 out of 43 samples tested were found to have levels of neoantigen corresponding to 2 to 11.5% complement activation, whereas 13 out of 14 normal donor plasmas contained amounts of neoantigen indicating much less than 1% complement activation.

Antibodies, Monoclonal↗
PubMed1985

Species specificity of recognition by the alternative pathway of complement.

The recognition function of the alternative complement pathway was studied with isolated human and rabbit components. Zymosan and homologous and heterologous erythrocytes were used as representative activators or nonactivators. The binding affinity of Factor B and Factor H for particle-bound C3b was measured. In both species, the average affinity of Factor H for bound C3b on homologous cells (nonactivators) was eight to 10 times higher than on zymosan particles (activators). The interaction between Factor H and C3b on rabbit erythrocytes was species-specific: rabbit Factor H bound strongly to rabbit C3b on rabbit erythrocytes and also on human erythrocytes, which are nonactivators for the rabbit alternative pathway. Human Factor H bound strongly to human C3b on human erythrocytes but seven times weaker on rabbit erythrocytes, which are activators of the human alternative pathway. No substantial differences were found in the binding of Factor B to bound C3b regardless of the nature of the particle to which C3b was bound. The results indicate that in the two species studied, the molecular mechanism of recognition is analogous and that recognition is species-specific.

Animals↗
PubMed1984

Molecular architecture of human properdin, a positive regulator of the alternative pathway of complement.

The structure of the human properdin molecule was investigated by hydrodynamic, spectroscopic, and transmission electron microscope studies. Sucrose density gradient ultracentrifugation of purified, functionally active properdin showed a single component sedimenting at 5.5 S. Electron microscopic examination by two different methods, however, revealed polydispersity of the protein which consisted of cyclic dimers, trimers, tetramers, pentamers, and higher cyclic oligomers. Approximately 80% of the oligomers were dimers, trimers, and tetramers. Monomers could not be detected. These polymers could be partially separated by gel filtration on Sephacryl S-300 and all fractions were active in terms of binding to C3b. The specific activity increased with oligomer size. When reexamined after incubation at 37 degrees C for 4 h or at 4 degrees C for 2 weeks, the chromatographic behavior of the oligomers and their electron microscopic appearance were unchanged, suggesting that in vitro no rapid equilibration occurred. The protomer is clearly visualized within polymers as a flexible, rod-like structure 26.0 nm in length and 2.5 nm in diameter, with pronounced thickening at each end. The monomer is bivalent with respect to binding to other properdin monomers and the binding sites are localized to the ends of the structure. A model could be devised which is consistent with the distinct geometry of the intersubunit contacts observed in micrographs. The circular dichroism spectrum of properdin suggests the presence of little alpha helix or beta structure and shows positive ellipticity at 231 nm. In contrast to previous investigators, we conclude that isolated human properdin is polydisperse and consists of a set of cyclic polymers constructed from a single highly asymmetric and flexible protomer.

Binding Sites↗
PubMed1984

Characterization of the initial C3 convertase of the alternative pathway of human complement.

C3(H2O),Bb, the initial C3 convertase of the alternative complement pathway, was demonstrated to be a metal-containing protein complex by sucrose density gradient ultracentrifugation. Demonstration of this labile enzyme became possible by increasing its half-life with nickel instead of magnesium ions used for enzyme formation. The enzyme was generated from C3, the internal thioester bond of which was hydrolyzed (C3(H2O)), and from 125I-Factor B and Factor D. The sedimentation coefficient of the enzyme complex was 10.7S. By using 63Ni for enzyme formation, the metal ion was detected in the enzyme complex after ultracentrifugation in the presence of 10 mM EDTA. The stoichiometry of the constituents in the C3(H2O),Bb(Ni) complex was 1:1:1. To verify that C3 is incorporated into the enzyme complex in the form of C3(H2O), the enzyme complex was adsorbed to anti-Factor B-Sepharose and subjected to decay-dissociation. Examination of the subsequently eluted protein by SDS gel electrophoresis under reducing conditions demonstrated the presence of an intact C3 alpha-chain. This work provides further evidence that a C3 convertase can be generated from noncleaved C3 that is modified at the thioester site. With the use of a fluorometric assay, the activity (kcat/Km) and the half-life of the initial C3 convertase were determined and compared to those of C3b,Bb.

Chemical Phenomena↗
PubMed1983

C3 convertase of the alternative complement pathway. Demonstration of an active, stable C3b, Bb (Ni) complex.

The purposes of this study were to demonstrate the C3 convertase complex, C3b, Bb (EC 3.4.21.47), of the alternative pathway of complement by ultracentrifugation and to determine whether the metal ion required for enzyme formation is present in the active enzyme complex. It has been shown previously that C3b,Bb formed with Ni2+ rather than Mg2+ exhibits enhanced stability. Using sucrose density gradient ultracentrifugation, an enzymatically active C3b,Bb(Ni) complex could be demonstrated which has a sedimentation coefficient of 10.7 S and which is stable in 10 mM EDTA. Upon formation of the enzyme with the radioisotope 63Ni2+, the ultracentrifugal distribution of the metal correlated with that of the enzyme complex. The molar ratio of Ni to C3b,Bb was 1:1. Displacement of Ni by Mg during formation of the enzyme indicated that both metals may bind to the same site in the enzyme. Binding of 63Ni to the catalytic site bearing fragment Bb was significantly stronger than its binding to C3b or to the zymogen, Factor B. It is proposed that there is one metal-binding site in the C3b,Bb enzyme which is not susceptible to chelation by EDTA and which is located in the Bb subunit.

Complement Activating Enzymes↗
PubMed1983

Paroxysmal nocturnal hemoglobinuria: deficiency in factor H-like functions of the abnormal erythrocytes.

Erythrocytes from patients with paroxysmal nocturnal hemoglobinuria (PNH) contained a subpopulation that lacked membrane-associated Factor H-like activity present on normal human erythrocytes. Initial deposition of C3b on the erythrocytes was effected using a fluid phase C3 convertase. The cells were then treated with fluorescein-labeled C3 and the cell-bound C3 convertase. Analysis utilizing the fluorescence-activated cell sorter revealed two distinct cell populations, one of which was highly fluorescent, indicating a large number of C3b molecules per cell. Only this population (43%) was susceptible to lysis (44%) when exposed to acidified serum before C3b deposition. The less fluorescent population resembled normal human erythrocytes and was not affected by prior treatment with acidified serum. Since C3b deposition occurred almost exclusively on the complement-sensitive cells in the PNH erythrocyte population, these cells could be examined for the Factor H-like regulatory activities without prior isolation. These functions include enhancement of inactivation of erythrocyte-bound C3b by Factor I and acceleration of the decay of erythrocyte-bound C3 convertase, C3b,Bb. It was found that C3b on PNH erythrocytes was 100-fold less susceptible to inactivation by Factor I than C3b on normal human erythrocytes. The half-life at 22 degrees C of C3b,Bb on PNH erythrocytes was threefold greater than on normal human erythrocytes and similar to that of the enzyme bound to particles that do not possess Factor H-like activity. These observations suggest that the abnormal susceptibility of PNH erythrocytes to lysis by complement is due to a functional deficiency in one or more of the Factor H-like proteins present on normal human erythrocytes.

Complement C3↗
PubMed1983

Deficiency of an erythrocyte membrane protein with complement regulatory activity in paroxysmal nocturnal hemoglobinuria.

Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired hemolytic anemia in which the erythrocytes are abnormally sensitive to lysis by complement. A functional deficiency of membrane-associated complement regulators has been demonstrated on PNH erythrocytes. The two factor H-like proteins, the C3b receptor (CR1) and the decay-accelerating factor (DAF), were isolated from normal human erythrocytes, and specific antisera were prepared. Selective inhibition of the two proteins on normal erythrocytes by the antisera demonstrated (i) that the factor responsible for accelerated decay of erythrocyte-bound C3 convertase is DAF and (ii) that the cofactor required for inactivation of erythrocyte-bound C3b by factor I is CR1. PNH erythrocytes were deficient in both of these activities. Erythrocytes deficient in CR1, which were obtained from an apparently healthy individual, exhibited normal DAF activity but no factor I cofactor activity. These cells were not susceptible to complement-mediated lysis in acidified human serum, whereas PNH erythrocytes and Pronase-treated human erythrocytes (which lack DAF and CR1 activities) were lysed by this treatment. It is suggested that the protein primarily responsible for preventing complement activation on normal human erythrocytes is DAF. AMr 73,000 protein isolated from the normal erythrocyte membranes of one PNH patient by using anti-DAF IgG was largely absent from the abnormal erythrocytes of this individual, suggesting that PNH cells lack the DAF protein. CR1 antigen, however, was present on the abnormal PNH erythrocytes. The results suggest that the primary molecular defect underlying the clinical manifestations of PNH may be the lack of the membrane-associated DAF protein and that the abnormal cells may also exhibit impaired CR1 function.

Complement Activating Enzymes↗