A comparative analysis of the C1-binding ability of fragments derived from complement-fixing and noncomplement-fixing IgM proteins.

The purpose of this study was to examine the molecular parameters necessary for initiation of complement fixation by IgM proteins. To determine why some IgM molecules are capable of complement fixation while others are not, several different Waldenström IgM proteins were examined for their ability to fix total hemolytic complement in the CH(50) assay. Subsequently, the C1 fixing ability of a 56-residue fragment derived from the Cmu4 domain of each of these IgM molecules was studied with C1 fixation assay. One of the three Waldenström IgM proteins (Gr) used in the present study was found unable to consume complement in a CH(50) assay when tested at the same concentration as the two complement-consuming IgM molecules (Dau and Bus). However, when the 56-residue C(H)4 fragment from the Cmu4 domain of each IgM molecule was tested for C1-fixing ability, all three were found to bind C1. On the basis of these observations, it is proposed that a C1 binding site exists within the Cmu4 domain of both complement-fixing and noncomplement-fixing IgM molecules. Presumably, the latter molecules are unable to interact in their native state with C1 in the manner required for initiation of the classical complement pathway, possibly due to the configurational inaccessibility of the entire C1 binding site.

IgM complex receptors on subpopulations of murine lymphocytes.

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)5mu but not by Fab mu. 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.

C1 fixation and classical complement pathway activation by a fragment of the Cmu4 domain of IgM.

A 56 residue fragment derived from a Waldenströme IgM protein and consisting of 24 residues of the amino-terminal portion of the Cmu4 domain disulfide bonded to 32 residues of the carboxy-terminal region of the loop has been shown to fix active C1 (C1) in a C1-fixation assay. Cleavage of the disulfide bond within the CH4 fragment resulted in a marked decrease of C1-fixing ability, although the isolated A and B fragments did retain a limited ability to fix C1. Upon incubation with normal human serum the intact CH4 fragment and equal molar amounts of the isolated A and B peptides consumed C4 suggesting that the C1-activating determinant of IgM remains intact in these three fragments. Furthermore, on a molar basis the intact or the reduced CH4 fragment consumed C4 as effectively as each of its component chains suggesting that transient binding of C1 by the individual A and B peptide chains is sufficient to activate C1. On the basis of these observations it is proposed that a classical complement fixation function, i.e. C1 binding and activation, can be localized within a region of the IgM molecule corresponding to the Cmu4 domain.

The structural basis for binding of complement by immunoglobulin M.

An insight into the structural features of human IgM that are responsible for its capacity to bind the first component of complement (C) has been obtained by examining the ability of IgM subfragments to bind active C1 (C1). The smallest two fragments found to bind C1 were the major CNBr fragment of the Fc portion of IgM and the C(H)4 fragment of the carboxy-terminal domain. The smallest fragment which fixes C1 has a disaggregated mol wt of 6,800, consists of 60 residues, and contains no carbohydrate. Structural considerations and sequence overlaps suggest that the amino-terminal side of the C(H)4 domain (24 amino acid residues) might be responsible for fixing C1.