The 28K and 70K dalton polypeptide components of mouse Ra—reactive factor are responsible for bactericidal activity

Ihara, Izumi; Ihara, Setsunosuke; Nagashima, Akiko; Ji, Yue-Hua; Kawakami, Michiyuki

doi:10.1016/s0006-291x(88)80086-x

Cited by 12 publications

(4 citation statements)

References 9 publications

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“…Mouse RaRF has been most characterized (28)(29)(30) and was found to consist of at least two components: a carbohydrate-binding component and a C4-and C2-consuming component. The former component (P28a and P28b) shares structural properties with MBP (31), and the latter is a protease that displays a Cls-like activity in the classical pathway activation by RaRF (32,33). We recently demonstrated that human MBP is a component of RaRF (19).…”

Section: Discussionmentioning

confidence: 99%

Activation of the classical complement pathway by mannose-binding protein in association with a novel C1s-like serine protease.

Matsushita

Fujita

1992

The Journal of Experimental Medicine

591

359

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SummarySerum mannose-binding protein (MBP) is a C-type lectin that binds to terminal mannose and N-acetylglucosamine moieties present on surfaces of certain pathogens and activates the classical complement pathway. In the present study, we describe the mechanism underlying the activation triggered by MBP. The human serum MBP fraction was obtained by sequential af~nity chromatography on mannan-Sepharose, anti-IgM-Sepharose and anti-MBP-Sepharose in the presence of calcium ions. This fraction contained a Cls-like serine protease as assessed by C4 consumption. The Cls-like serine protease, designated MBP-associated serine protease (MASP), was separated from MBP by rechromatography on anti-MBP-Sepharose in the presence of ethylenediaminetetraacetic acid. MASP exhibited both C4-and C2-consuming activities. The molecular mass of MASP was estimated to be 83 kD with two polypeptides of heavy (66 kD) and light (L) (31 kD) chains linked by disulfide bonds. The serine residue responsible for protease activity is located on the L chain. Reconstitution experiments using MASP and MBP revealed that combination of the two components restores C4-and C2-activating capacity on mannan. Based on analyses of mdecular size, antigenicity, and 11 NH2-terminal amino acid sequences of the L chain, we conclude that MASP is a novel protein different from Clr or Cls. Our findings are not in accord with a proposed mechanism by which MBP utilizes the Clr2-Cls2 complex to initiate the classical complement pathway.

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Section: Discussionmentioning

confidence: 99%

Activation of the classical complement pathway by mannose-binding protein in association with a novel C1s-like serine protease.

Matsushita

Fujita

1992

The Journal of Experimental Medicine

591

359

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“…In fact, not even the outermost portions of core polysaccharides appear to be unmasked by normal serum, since exposure of the Ra chemotype renders wild-type bacteria susceptible to the bactericidal activity of complement. 102,103 Furthermore, the serum-enzyme-unmasking hypothesis has been directly tested and not substantiated. Gigliotti and Shenep, using non-boiled organisms, studied binding of four J5 MAbs to the J5 rough mutant and to two strains of smooth E, coli (parental 0111:B4 and 07:K1), before and after 30 min of incubation in guinea pig serum with complement.…”

Section: Rough-mutant Vaccines: Historical Perspectivementioning

confidence: 99%

Review: Evidence against the hypothesis that antibodies to the inner core of lipopolysaccharides in antisera raised by immunization with enterobacterial deep-rough mutants confer broad-spectrum protection during Gram-negative bacterial sepsis

Greisman

Johnston

1997

Journal of Endotoxin Research

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Antisera to rough enterobacterial mutants of chemotypes Ra, Rc, and Re have been reported to confer broad-spectrum protection against wild-type smooth strains. It has been hypothesized that binding and neutralization of lipopolysaccharides (LPS) by antibodies to common core epitopes underlies such protection. This review summarizes experiments by our laboratory and others that do not confirm this concept and proposes reasons for the divergent results. Studies indicating broad-spectrum protection by rough-mutant antisera often had defects in experimental design or methodology. These include the failure: (i) to use matched pre- and postimmune sera from the same donors to control for variable protective activity of normal sera; (ii) to exclude the role of natural and polyclonally stimulated antibodies with proven protective activity against the infecting bacterial strain (e.g. O-specific, capsular, Pseudomonas exotoxin A); (iii) to exclude protective effects of acute-phase serum factors; (iv) to exclude protective effects of endotoxin contamination after adsorption or fractionation of antibody preparations; (v) to use non-boiled bacteria and LPS not subjected to acid-hydrolysis or gel-fractionation, and to exclude nonspecific adsorption, to demonstrate physiologically meaningful binding of rough-mutant antibodies to smooth enterobacteria and their LPS.

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“…Therefore, a polypeptide component(s) other than the P28 polypeptides plays (play) a role in the activation of C4 and C2. Probably, the 29-and 70-kDa polypeptides in the low-molecular-weight component are responsible for the C4/C2 activation (Ihara et al, 1988(Ihara et al, , 1991. Taking these results into consideration, we propose that RaRF is a complex composed of the C4activating proteases, together with the P28a and P28b.…”

Section: P28bmentioning

confidence: 99%

cDNAs and deduced amino acid sequences of subunits in the binding component of mouse bactericidal factor, Ra-reactive factor: similarity to mannose-binding proteins

Kuge¹,

Ihara²,

Watanabe³

et al. 1992

Biochemistry

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The complement-dependent bactericidal factor, Ra-reactive factor, binds specifically to Ra polysaccharide, which is common to some strains of Gram-negative enterobacteria, and its is a complex of proteins composed of a polysaccharide-binding component and a component that is presumably responsible for the complement activation. The former component consists of two different 28-kDa polypeptides, P28a and P28b. We determined the partial amino acid sequences of P28a and P28b, and the results indicated that these polypeptides were similar to two species of mannose-binding protein, MBP-C and MBP-A (alternative names, liver and serum mannan-binding proteins, respectively), which have been isolated from rat liver and/or serum [Drickamer, K., Dordal, M. S., & Reynolds, L. (1986) J. Biol. Chem. 261, 6878-6887; Oka, S., Itoh, N., Kawasaki, T., & Yamashina, I. (1987) J. Biochem. 101, 135-144]. Thus, we cloned the respective cDNAs, using as probes synthetic oligonucleotides for which the sequences had been deduced from the amino acid sequences of P28a and P28b and of rat MBP cDNAs. The primary structures of P28a and P28b deduced from the cloned cDNAs are homologous to one another. They have three domains, a short NH2-terminal domain, a collagen-like domain, and a domain homologous to regions of some carbohydrate-binding proteins, as has been reported for rat MBPs. Southern and Northern blotting analyses using these cDNAs indicated that the P28a and P28b polypeptides are the products of two unique mouse genes which are expressed in hepatic cells.

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The 28K and 70K dalton polypeptide components of mouse Ra—reactive factor are responsible for bactericidal activity

Cited by 12 publications

References 9 publications

Activation of the classical complement pathway by mannose-binding protein in association with a novel C1s-like serine protease.

Activation of the classical complement pathway by mannose-binding protein in association with a novel C1s-like serine protease.

Review: Evidence against the hypothesis that antibodies to the inner core of lipopolysaccharides in antisera raised by immunization with enterobacterial deep-rough mutants confer broad-spectrum protection during Gram-negative bacterial sepsis

cDNAs and deduced amino acid sequences of subunits in the binding component of mouse bactericidal factor, Ra-reactive factor: similarity to mannose-binding proteins

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