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.

An insight into the structural features of human Waldenstr6m IgM proteins which are responsible for their capacity to bind and activate the first component of complement (C) has been obtained . The basic approach has involved enzymatic and chemical fragmentation of the IgM molecule followed by examination of the resulting fragments for C-fixing activity . A 6,800 mol wt fragment obtained from the terminal C/,4 domain of a Waldenstr6m IgM molecule has been shown to bind active C1 (C1) (1). This subfragment of IgM, which was designated the C,,4 fragment, was found to be composed of the CHc4 domain of the Fcp, minus two tryptic peptides in the center of the loop . The first set of experiments described in this communication were designed to evaluate the dependence of C1-binding ability upon the presence of the intrachain disulfide bond in the C,,4 fragment. In addition, the CH4 fragment before and after reduction, as well as each of its component chains, were examined for their ability to trigger the activation of the classical C pathway. Based upon these studies we are able to present evidence suggesting that the C1-activating function remains intact in these three fragments. Materials and MethodsPreparation of IgM and Its C 4 Fragment . The methods used to isolate IgM and to obtain the CH4 fragment have been previously reported (1, 2) .Isolation of the Two Peptide Chains Comprising the C 4 Fragment . C H4 fragments (5 mg/ml in 5 M guanidine-HCI adjusted to pH 7.5 by Tris base) were reduced by the addition of 2-mercaptoethanol to a final concentration of 0.1 M. After 3 h of incubation at room temperature, a 10% excess of iodoacetamide was added and the pH was maintained at 7.5 by the addition of Tris base until alkylation was complete . The reduction mixture was desalted by passage through a Bio-Gel P-2 column (Bio-Rad Laboratories, Richmond, Calif.) eluted with 1% NH QHCO3 and lyophilized. Fractionation of the two peptides comprising the CH4 fragment was achieved by chromatography through a column of Whatman DE-52 cellulose (H . Reeve Angel & Co ., Inc., Clifton, N. J.) equilibrated with 0.025 M NH,HC0 3, pH 8.0 . After application of the sample, the column was washed with one to two total column vol of 0.025 M NH,HC03, pH 8.0, and a linear gradient (0 .025 M NH,HCO 3-1 M NH,HCO 3) was begun. Pooled fractions from each of the two peaks eluted from this column were lyophilized and redissolved in dextrose gelatin veronal buffer containing *

No abstract

Murine lymphocytes from spleen, lymph node, and thymus were examined for IgM complex receptors. Lymphocytes from all three organs were found to bind SRBC sensitized with IgM from various sources including: primary anti-SRBC serum, murine and rabbit anti-Escherichia coli LPS sera, and a murine IgM myeloma (MOPC 104E). Rosette formation by lymphocytes with IgM-sensitized SRBC was inhibited by soluble antigen-IgM complexes but not by IgM or antigen alone. Rosette formation was also inhibited by human IgM (Fc)5µ but not by Fab µ. Antiserum and complement treatment of the cells and subsequent recovery of the viable cells by trypsinization, filtration, and washing revealed the IgM rosette-forming cell (RFC) in the thymus to be a T cell. Spleen on the other hand was found to contain both B and T cells capable of binding IgM sensitized SRBC. Removal of both B and T cells from spleen cell suspensions eliminated all IgM RFC. The IgM complex receptor was found to be trypsin insensitive. Anti-Ig column fractionation enriched IgM RFC in spleen and lymph node suspensions passed through the columns, whereas cells bearing surface Ig, IgG complex receptors, and C3 receptors were retained in the columns.