Complement Fixing and Macrophage Opsonizing Activities Associated with β2 Microglobulin

Painter, R. H.; Yasmeen, D.; Assimeh, S. N.; Poulik, M. D.

doi:10.3109/08820137409055743

Cited by 10 publications

(9 citation statements)

References 2 publications

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“…The intact protein, in contrast, may be renatured from high concentrations of guanidine without difficulty. The change in spectral properties of reduced f32-microglobulin under conditions where the disulfide is allowed to reform indicate that the native conformation can be substantially recovered.t * This derivative also inactivated whole complement, as measured in a classical complement fixation test, as has already been reported for the native protein (9). The relative potencies of the two preparations appear to be equal.…”

Section: Resultsmentioning

confidence: 54%

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The structure and function of immunoglobulin domains: studies with beta-2-microglobulin on the role of the intrachain disulfide bond.

Isenman¹,

Painter²,

Dorrington³

1975

Proc. Natl. Acad. Sci. U.S.A.

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fl-Microglobulin, a low-molecular-weight protein structurally related to the homology regions of immunoglobulins, has been used to study the role of the intrachain disulfide bond in the unfolding of immunoglobulin domains. The intact protein could be reversibly unfolded in guanidine hydrochloride, as judged by circular dichroism and optical rotation. Similarly, reoxidation of the reduced protein, during transfer from high concentrations of guanidine to neutral aqueous buffer, yielded a product with spectral characteristics typical of the native protein. However, if the free SH groups were prevented from reoxidizing either by chemical modification or by holding them in the reduced state, the molecule appeared to be in the randomly coiled state even under conditions where the intact protein is in the native conformation, judged on the basis of chiroptical measurements. The complement-fixing activity exhibited by native #2-microglobulin was retained by the reduced and alkylated derivative, suggesting that the site may be formed from a linear array of amino acids. We suggest a model for the folding of 02-microglobulin (and immunoglobulin domains) in which one of the early folding events results in disulfide bond formation, the latter being an obligatory step for continued folding to the native state.Extensive amino-acid sequence data on kappa and lambda light (L) chains and heavy (H) chains from several immunoglobulin classes in a variety of animal species, indicate that these chains are divided into nonoverlapping homology regions (1). Each region comprises some 110 residues and contains a single intrachain disulfide bond. On the basis of this information and a variety of observations regarding the limited susceptibility of immunoglobulin subunits to proteolytic cleavage, Edelman et al. (2) The absolute conservation of the two half-cystines contributing to the intrachain disulfide bond suggests that this bond plays an important role within the domain. In an earlier study, Lapanje and Dorrington (4) found that extensively reduced and alkylated IgG behaved as a randomly coiled protein even in low concentrations of guanidine hydrochloride (Gdn. HCl) where the intact protein was in the native state. This observation suggested that the formation of intrachain disulfides is an obligatory step during the refolding of IgG, since if these bonds were left intact the native state of the IgG (as judged by optical rotation) was recovered when the guanidine was removed.The possible significance of the above observation with respect to the acquisition of the three-dimensional structure of IgG during biosynthesis prompted us to seek a simpler system in which to study domain folding. 032-microglobulin consists of a single polypeptide chain of 100 residues (molecular weight, 11,700), and sequence analysis (5, 6) has shown that the protein is homologous to the constant (C) homology regions of IgG (i.e., CL, C,,1, CA2, and CI3). A single disulfide bond is present, enclosing a loop of residues of similar size to that found i...

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Section: Resultsmentioning

confidence: 54%

“…The direct interaction of native and reduced/alkylated 02-microglobulin with the first component of complement was measured using the method developed by Augener et al (14) with previously described modifications (9).…”

mentioning

confidence: 99%

The structure and function of immunoglobulin domains: studies with beta-2-microglobulin on the role of the intrachain disulfide bond.

Isenman¹,

Painter²,

Dorrington³

1975

Proc. Natl. Acad. Sci. U.S.A.

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“…16 for reactions with macrophages). It was found that at high concentrations p2-p and the Fc portion of human IgG (aggregated and unaggregated) moderately inhibited EA rosette formation (Fig.…”

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confidence: 98%

Evidence for Clustering of H-2K, H-2D, and the Fc Receptor on the Membranes of B Cells

Tyan

1976

Experimental Biology and Medicine

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“…It should be noted that the 24 residue sequence common to CH4 and CNBr 5 has the highest degree of homology to human /~2-microglobulin of any in the terminal domain, having 8 out of 24 identities and a total of 14 out of 24 conservative replacements (see Putnam et al [10] and Peterson et al [12] for comparative sequences). The relevance of this is pointed out by the fact that Painter et al (13) have shown that/~2-microglobulin fixes CT with high efficiency. On the other hand, a similar sequence comparison to the IgG domains (14) reveals that the 24 residues of CH4 have a higher degree of homology to C~3 than to the C~,2 domain which has been suggested to be responsible for C activation on the basis of data accumulated using the CHs0 assay (2,3).…”

Section: Discussionmentioning

confidence: 93%

The Structural Basis for Binding of Complement by Immunoglobulin M

Hurst

Volanakis

Hester

et al. 1974

The Journal of Experimental Medicine

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Knowledge of antibody structure is now sufficient to allow investigation of problems of biological function relative to structural parameters. One such structure-function relationship that can be approached is the molecular basis for complement (C) activation by immunoglobulins. On the basis of structural studies it has been proposed that the homology regions of immunoglobulins are arranged in a series of compact domains, with each domain mediating a biological function (1). In line with this hypothesis it has been suggested that the C~,2 domain may play a role in C fixation by IgG (2, 3). In 1971 Augener et al. by use of active C1 (CT) fixation assay showed that the 7S subunit of IgM could bind CT (4). Plaut et al. subsequently demonstrated that the (Fc)stt and Fctt fragments of IgM were capable of fixing C (5). We have investigated the relative capacity of small molecular weight fragments of a human WaldenstrSm IgM protein (Dau) to bind the first component of C and are thus able to present evidence for the localization of this function to a 24 amino acid sequence. Materials and MethodsPreparation of IgM and Its Subfragments. IgM was isolated from the plasma of a patient (Dau) with Waldenstr6m's macroglobulinemia as previously described (6). (Fc)stt and FabtL fragments were prepared using trypsin digestion at 60°C (6). After separation of (Fc)5# and Fabtt on Sephadex G-200 in 1% NH4HCOs, pH 8, the (Fc)stt was further purified on Bio-Gel A-15 (Bio-Rad Laboratories, Richmond, Calif.). Cyanogen bromide (CNBr) cleavage of (Fc)~tz and Fabu was performed as described by Zikan and Bennett (7). Dau Fc~ is composed of four CNBr fragments (7), designated CNBr 5 through CNBr 8 (Fig. 1). After CNBr cleavage the smaller fragments, CNBr 6 and CNBr 8, were removed by gel filtration (7). The disulfide bonds in the pentameric CNBr 5 plus 7 fragment were then gently cleaved (either by partial oxidative sulfitolysis [8] or using 0.01 M dithiothreitol followed by alkylation with iodoacetamide) and the sample was applied to a Sephadex G-150 column in 5 M guanidine-HC1 to obtain CNBr 5 dimers and monomers which were free of CNBr 7, as shown by polyacrylamide disc electrophoresis in sodium dodecyl sulfate.In addition, 33,000 mol wt Fctt fragments were prepared by reducing Fcstt with 0,05 M cysteine. The limited tryptic cleavage method of Hester and Schrohenloher (9) was used to release from the Fct~ fragments a homogeneous 6,800 mol wt fragment. This fragment was designated the C H4 fragment, since a comparison of its amino acid composition and partial sequence as determined in the sequenceP with that published by Putnam et al. (10) for the IgM, Ou, revealed that it was composed of the Cu4 domain of the FctL minus two tryptic peptides in the center of the loop (residues 468-491 and 515-550 according to the numbering of Ou). Relative to Dau CNBr fragments, C ~4 consisted of the carboxy-terminal 22 residues of CNBr 5 and the first two residues of CNBr 6 disulfide bonded to 36 residues of CNBr 7.

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Complement Fixing and Macrophage Opsonizing Activities Associated with β2 Microglobulin

Cited by 10 publications

References 2 publications

The structure and function of immunoglobulin domains: studies with beta-2-microglobulin on the role of the intrachain disulfide bond.

The structure and function of immunoglobulin domains: studies with beta-2-microglobulin on the role of the intrachain disulfide bond.

Evidence for Clustering of H-2K, H-2D, and the Fc Receptor on the Membranes of B Cells

The Structural Basis for Binding of Complement by Immunoglobulin M

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