Site of J Chain Attachment to Human Polymeric IgA

Abstract

A fragment containing J chain was released from human polymeric myeloma IgA protein by cyanogen bromide cleavage. The identity of the fragment was determined by its electrophoretic mobility and antigenic determinants. After purification by gel filtrations and DEAE-Sephadex chromatography, this fraction appeared similar (with respect to its amino acid and carbohydrate compositions and its peptide maps) to the J chain isolated from this IgA protein; the molecular weight was 17,000 i 100. Upon reduction and alkylation, with subsequent separation of peptides by gel filtration, three components were obtained: the largest component (molecular weight 13,400) corresponded to the N-terminal segment of J chain and contained a homoserine residue, the second corresponded to the C-terminal part of J chain with 13-18 amino acid residues, and the third corresponded to the C-terminal octapeptide of the a chain. The data indicate that J chain is attached to a chain(s) through the penultimate cysteine residue of the C-terminal octapeptide.In addition to heavy and light chains, human and animal polymeric immunoglobulins contain a polypeptide termed J chain (1-7) with a molecular weight of 15,600 4-200 (5, 8, 9). Studies of immunofluorescence and biosynthesis indicated that J chain was produced in plasma cells that synthesized IgA and IgM molecules (10-12). On the basis of the absence of J chain in monomeric and its presence in all polymeric immunoglobulins examined, its relatively large content of cysteine residues, and its disulfide bond attachment to immunoglobulin molecules, it was suggested that J chain joins the monomeric units of IgA and IgM to form, in an undefined manner, the respective polymeric molecules (13). Although the precise location of the disulfide linkages of J chain to IgA and IgM molecules has not been elucidated, studies on the products of proteolysis of these immunoglobulins strongly suggested an attachment in the Fc region of the heavy chain; the Fab and F(ab)2 fragments were devoid of J chain (14-16).It has been shown that J chain contains one methionine residue near the carboxy terminus (17). Therefore, part of the J chain linked by disulfide bonds to one or more heavy chain fragments should be present after cleavage with CNBr. This approach was applied for identification of the a-chain portion involved in the binding of J chain in a polymeric myeloma IgA. In this communication we present evidence which indicates that J chain is linked to the penultimate cysteine residue of the a chain(s). MATERIAL AND METHODSPurification and Characteristics of Polymeric IgA. Blood plasma from a patient (Fel) with IgA multiple myeloma was recalcified to remove fibrinogen, and a crude gamma globulin fraction was obtained by precipitation with ammonium sulfate to 50% saturation. The precipitate was collected by centrifugation and dialyzed against saline buffered with Tris -HCl at pH 7.4 and subsequently gel filtered through Sephadex G-200 and Sepharose 6-B as described in detail in a previous communication (18). The...

Mucosal Immunoglobulins

“…The ability of IgA and IgM to form polymers has been associated with the presence of the 18-amino-acid C terminal tailpieces on α and μ chains, which include the penultimate Cys residue to which J chain is attached (Mestecky et al, 1974b;Mestecky and Schrohenloher, 1974). Indeed, the addition of this tailpiece to IgG results in the formation of pIgG with interesting functional consequences (Sørensen et al, 1996;Smith et al, 1995).…”

“…Subsequent studies revealed that this chain was glycosylated, with a molecular mass of 15-16 kDa, and was linked by disulfide bridges to the Fc fragment of polymeric IgM or IgA of secretory and serum (myeloma) origin (Mestecky and Schrohenloher, 1974;Mestecky et al, 1974b;Garcia-Pardo et al, 1981). On the basis of the given criteria (molecular mass, fast electrophoretic mobility, characteristic amino acid composition, and immunochemical cross-reactivity), the presence of J chain has been established, with varying degrees of confidence, in pIg from many vertebrate species including mammals (human, monkey, rabbit, mouse, pig, dog, goat, cat, cow, horse, rat, guinea pig, and sheep), birds (chicken and pheasant), reptiles, (two species of turtles, Trachemis scripta, and Pelodiscus sinensis), amphibians (frog and toad), and fishes (catfish and nurse sharks) (Kobayashi et al, 1973;Inman and Mestecky, 1974;Iwata et al, 2002;Koshland, 1985;Mikoryak et al, 1988;Kulseth and Rogne, 1994;Takahashi et al, 2000;Hohman et al, 1997Hohman et al, , 2003Xu et al, 2009).…”

“…This high degree of primary structure homologies between IgA and IgM appears to reflect their close evolutionary origin. Furthermore, IgA and IgM molecules share important structure-function similarities such as the ability to form polymers, bind J chain through their penultimate Cys residues in structurally analogous tailpieces (Mestecky et al, 1974b;Mestecky and Schrohenloher, 1974), and interact with epithelial pIgR essential for the selective transepithelial transport of pIg (Brandtzaeg et al, 1981(Brandtzaeg et al, , 1985. Moreover, S-IgM functionally replaces S-IgA in most IgAdeficient individuals (Plebani et al, 1983;Arnold et al, 1977;Brandtzaeg et al, 1968Brandtzaeg et al, , 1999.…”

“…J chains are generally rich in acidic (Asp, Glu) amino acid residues and low in Gly, Ser, and Phe; Trp tends to be absent (Inman and Mestecky, 1974). Six of the eight Cys residues form three intrachain disulfide bridges (Cys 12-Cys 100, Cys 17-Cys 91, and Cys 108-Cys 133); the other two residues (Cys 14 and Cys 68) are involved in disulfide bonds that link J chain to the penultimate cysteine residues of α-and μ-chains (Mestecky et al, 1974b;Mestecky and Schrohenloher, 1974;Frutiger et al, 1992;Bastian et al, 1992;Krugmann et al, 1997). Studies of the nurse shark J chain revealed several unexpected findings.…”

Parallel synthesis of immunoglobulins and J chain in pokeweed mitogen-stimulated normal cells and in lymphoblastoid cell lines.

“…J chain was isolated from an IgA2~, polymemc myeloma protein (8) and coupled to BSA as described by Brandtzaeg (5). Rabbits were immunized three times, at 1-mo intervals, with the antigen m complete Freund's adjuvant.…”