Assembly and secretion of heavy chains that do not associate posttranslationally with immunoglobulin heavy chain-binding protein.

Abstract

Abstract. Heavy chain-binding protein (BiP) associates posttranslationally with nascent Ig heavy chains in the endoplasmic reticulum (ER) and remains associated with these heavy chains until they assemble with light chains. The heavy chain-BiP complex can i be precipitated by antibody reagents against either component. To identify sites on heavy chain molecules that are important for association with BiP, we have examined 30 mouse myelomas and hybridomas that synthesize Ig heavy chains with well characterized deletions. Mutant Ig heavy chains that lack the C,1 domain could not be demonstrated to associate with BiP, whereas mutant Ig heavy chains with deletions of the CH2 or CH3 domain were still able to associate with BiP. In two light chain negative cell lines that produced heavy chains with deletions of the CH1 domain, free heavy chains were secreted. When Ig assembly and secretion were examined in mutants that did not associate with BiP, and were compared with normal parental lines, it was found that the rate of Ig secretion was increased in the mutant lines and that the Ig molecules were secreted in various stages of assembly. In one mutant line (CH1-) approximately one-third of the secreted Ig molecules were incompletely assembled, whereas the Ig molecules secreted by the parental line were completely assembled. Our data show the CH1 domain to be important for association with BiP and that when this association does not occur, incompletely assembled heavy chains can be secreted. This implies a role for BiP in preventing the transport of unassembled Ig molecules from the ER.I MMUNOGLOBULIN biosynthesis, assembly, and transport have been well characterized using both normal and malignant lymphoid cells (7,27). The Ig molecule is composed of two identical heavy chain proteins and two identical light chain (LC) t proteins which are joined by interchain disulfide bonds. Nascent heavy and light chains contain a hydrophobic amino-terminal signal sequence which allows them to be cotranslationally translocated into the lumen of the endoplasmic reticulum (ER) (5). The addition of mannose core sugars to heavy chains occurs during this translocation (4). Very soon after translocation, heavy and light chains begin to assemble in the ER and interchain disulfide bonds are formed (24,27). The sequence of subunit assembly appears to be determined by the heavy chain isotype. IgM and some IgG2b assemble as heavy and light chain (HL) molecules and then H2~ molecules, whereas IgGt and IgGz, first form H2 molecules, then H2L, and finally intact H2L2 molecules (3). Once assembly is complete, the Ig molecule is transported to the Golgi complex for further processing of 1. Abbreviations used in this paper: BiP, heavy chain-binding protein; ER, endoplasmic reticulum; LC, light chain; HCD, lymphoproliferative heavy chain disease; HL, heavy and light chain. its carbohydrate side chains (14). The mechanism for directing the Ig molecule to Golgi is not well understood. Data gathered from studying the transport of other proteins to the G...

Mutation of the ER retention receptor KDELR1 leads to cell-intrinsic lymphopenia and a failure to control chronic viral infection

“…BiP itself was discovered as a heavy chain binding protein (1) and associates with the C H 1 domain until replaced by correctly folded light chains (24). The fact that IgM expression is not impaired suggests that KDELR1 has different effects upon TCR versus BCR surface expression and that other KDELR1-dependent pathways may be important for promoting B-cell development.…”

“…Fifty microliters of blood was subjected to two rounds of red blood cell lysis with ammonium chloride before staining. Lymphocyte suspensions from blood, bone marrow (femurs and tibias from one hind leg), spleen, and thymus were counted (Z2 Coulter Counter; Beckman Coulter) and stained with a combination of the following mouse-reactive antibodies: FITCconjugated IgM (goat polyclonal; 1020-02; Southern Biotech); FITC-conjugated F4/80 (BM8); PE-conjugated IgD (11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25)(26); PerCP-Cy5.5-conjugated TCRβ (H57-597); APC-conjugated CD8α (53-6.7), CD11b (M1/70), CD93 (AA4.1), IgM (II/41), Ly6G (RB6-8C5), NK1.1 (PK136), TER119 (TER-119; eBioscience); FITC-conjugated CD4 (GK1.5), CD23 (B3B4), HY-TCR (T3.70), PE-conjugated CD11b (M1/70), CD19 (1D3), CD21/35 (7G6), CD44 (IM7), γδ TCR (GL3), Vα2 TCR (B20.1), PerCP-Cy5.5-conjugated B220 (RA3-6B2), CD8α (53-6.7), CD19 (1D3), NK1.1 (PK136); CD44 (IM7; Horizon V500; BD); APC-conjugated CD3e (145-2C11); APCCy7-conjugated CD45.2 (104), PE-Cy7-conjugated CD19 (6D5), CD45.1 (A20; Pacific blue), and CD16/32 (93; purified; BioLegend). 7-Aminoactinomycin D (7-AAD) was purchased from eBioscience, and EdU labeling and staining were performed according to the manufacturer's instructions (Invitrogen) 4 h after a single i.p.…”

The structural analysis of shark IgNAR antibodies reveals evolutionary principles of immunoglobulins

“…Similarly, the C H 1 domain of IgG, which does not possess the characteristic sequence elements of intrinsically disordered proteins (41), is unfolded in isolation (33). It plays a critical role in IgG assembly control (33,35,42), and analogously, C5 folding might depend on assembly of the complete IgNAR chains. Alternatively, authentic glycosylation or extrinsic cellular factors might be required for C5 folding.…”

Immunoglobulin gamma 2b transgenes inhibit heavy chain gene rearrangement, but cannot promote B cell development.

“…The association between X5 and j. heavy chains seems to occur through the CH1 domain of it, as deletions of this domain result in loss of association between /,4 and these polypeptides (43). Based on the conservation ofBiP binding and light chain binding to y2b via associations within the CH1 domain, it seems reasonable to expect that X5, which is structurally similar to a light chain, may also associate with y2b (44)(45)(46)(47) . However, even if y2b can bind X5, a correct signal for B cell maturation may not be created.…”