Association of transport-defective light chains with immunoglobulin heavy chain binding protein

Immunoglobulin light chains are usually secreted from cells when they are synthesized alone or in molar excess of heavy chains, but, there have been reports of nonsecreted light chains. We wished to determine whether immunoglobulin heavy chain binding protein (BiP), which blocks the transport of free heavy chains, might be responsible for the lack of secretion of some light chains. In two murine lymphoid cell lines that synthesize but do not secrete immunoglobulin light chains, the free light chain polymers were found bound to BiP. Examination of 20 other cell lines and hybridomas failed to disclose any cells synthesizing free or excess light chains that associated with BiP, in all cases the free light chains were secreted as dimers. Despite their association with BiP and their blocked secretion, the aberrant light chains could combine with heavy chains and could be secreted as intact Ig molecules. Thus, while light chains do not usually express signals which allow them to bind to BiP, it appears that such signals can be expressed on certain light chains, resulting in their combination with BiP and blocked secretion. When single chain mutant cell lines are isolated from parental lines producing both heavy and light chains, they are almost always light chain producers suggesting that free heavy chains are much more toxic than free light chains. In both PC700 and P3X63Ag cells, however, clones that have lost either heavy chains or transport-defective light chains are present at the same frequency. Our findings that the light chains in both of these lines are associated with BiP raise the possibility that BiP actually contributes to heavy chain toxicity instead of preventing it.     

Interaction of heavy and light chains of myeloma immunoglobulin with pooled normal chains: quantitative analysis of association and restoration of idiotype

We have analyzed in vitro recombinants between the isolated heavy (H) or light (L) chains of mouse myeloma protein MOPC-21 and L or H chains of normal mouse serum immunoglobulin (Ig). In the first series of experiments using fixed H chain and solubilized L chain, we have found out that only about 30% of normal L chain pool interact efficiently with individual H chain. Moreover, fractions with different affinity to H-MOPC-21 appeared to exist among normal L chains. In the second set of experiments recombination of H and L chains in solution was used. Examination of recombinants between myeloma H chain and normal L chains revealed a set representing 6% of L chain repertoire capable of forming MOPC-21-like idiotypic structure.     

The VKIIIb light chain sub-subgroup: restricted association with mu heavy chain in normal human serum.

The VKIII human kappa light chain subgroup has been serologically and structurally divided into two sub-subgroups, VKIIIa and VKIIIb. VKIIIb has been shown by others to be strikingly prevalent in IgM autoantibodies, but no studies have been performed to determine heavy chain isotype association with VKIIIb light chain in normal human serum. The VKIIIb sub-subgroup was shown here to be associated with mu heavy chain in normal human serum, but was not detected in association with gamma or alpha heavy chain. Approximately 25 +/- 15% of IgM-kappa was determined to be VKIIIb. Both intact IgG and purified light chains from pooled IgG did not bind monoclonal anti-VKIIIb, indicating that the determinants recognized by anti-VKIIIb are not merely masked in intact IgG. These results are the first report of a light chain sub-subgroup showing preferential association with a heavy chain isotype.     

Characterization of axolotl heavy and light immunoglobulin chains by monoclonal antibodies

Axolotl specific antibodies to 2,4-dinitrophenyl (DNP) were purified by affinity chromatography from the sera of animals immunized with 2,4,6-trinitrophenylated sheep red blood cells (TNP-SRBC). The purified anti-TNP/DNP antibodies, when analyzed by SDS-PAGE, were constituted of high molecular weight molecules, which in reducing conditions, were separated into heavy 72-88 kD and light 27-30 kD polypeptides. The axolotl heavy antibody chains strongly bound Concanavalin-A and migrate faster in SDS-PAGE after endoglycosidase-F (Endo-F) treatment. Using the same techniques, no carbohydrate components were detected onto light chains. Monoclonal antibodies (MAbs) were obtained against these purified axolotl immunoglobulins (Ig) and their specificities were studied by immunoblotting. MAbs 33.45.1 and 33.101.2 respectively recognized heavy and light chains determinants of the Ig molecule. These determinants were resistant to Endo-F digestion, suggesting that the two MAbs were not directed to polypeptide-associated N-linked high mannose or complex oligosaccharides. MAbs 33.45.1 and 33.101.2 were compared to 11.5.2, an anti-axolotl thymocytes MAb which was reactive for both axolotl leucocytes and soluble Ig. MAb 11.5.2 reacted in immunoblotting against several high molecular weight axolotl serum proteins, including heavy Ig chains. Light chains were not recognized. However, 11.5.2 did not further recognize Endo-F treated Ig, suggesting its specificity for a carbohydrate determinant of the heavy chain, and link to a large diversity of soluble or membrane glycoproteins.     

Co-deposition of amyloidogenic immunoglobulin light and heavy chains in localized pulmonary amyloidosis

Localized pulmonary amyloidosis is a rare condition whose pathogenesis is insufficiently understood. In the present study, we report a case of localized pulmonary amyloidosis associated with lung-restricted lymphoplasmacytoid lymphoma, monoclonal for immunoglobulin (Ig) G lambda (λ). Biochemical microtechniques have been applied for extraction, purification, and characterization of amyloid proteins. Surprisingly, chemical analysis of these proteins revealed a not-previously-described case of combined deposits containing Ig fragments of gamma heavy chain (variable domain) and λ light chain (constant domain). In view of the absence of circulating monoclonal Ig, this case supports the hypothesis that localized amyloid is formed by local plasmacytoid cells.     

Hybrid 7 S immunoglobulin molecules formes in vivo by association of heavy and light chains of different parental origin

Rabbits were immunized with the purified 7 S immunoglobulins of normal domestic cock (Ga), guinea hen (Nu) and their hybrid, the Numigall (NuGa). The antisera rendered specific for Ga and Nu immunoglobulins by appropriate absorptions were used to study the antigenic structure of the NuGa 7 S immunoglobulins. Nine to twenty percent of the NuGa molecules showed only Ga antigenic determinants, and six to twelve percent only Nu antigenic determinants. More than half of the NuGa immunoglobulins presented both Nu and Ga antigenic determinants on the same molecule. The heavy and light chains of the 7 S immunoglobulins of the three species were isolated and studied with the same rabbit antisera. The isolated chains of the NuGa immunoglobulins showed either the antigenic structure of Nu or Ga. No hybrid chains could be demonstrated. A model of in vivo chain assembly of heavy and light chains from different parental origin is proposed to explain the presence of hybrid molecules in the NuGa 7 S immunoglobulins.     

Recombination of heavy and light chains from human immunoglobulins

The recombination of reduced, alkylated heavy and light chains from three IgG, two IgM and one IgA pathological protein and from normal IgG has been quantitatively studied.

All γ, α and μ chains showed some recombination with all κ and λ chains tested. One of the IgG and two of the IgM proteins showed more than 50 per cent recombination and in these there was evidence of a selective reconstitution of autologous chain pairs; the structural features which determine re-association appear to be independent of the electrophoretic mobility, or antigenic and allotypic specificity of the interacting chains. The chains of normal IgG and of three myeloma proteins showed a low percentage recombination and these autologous chain pairs did not combine preferentially. It appears that immunoglobulin chains show a variable ability to regain their native configuration after isolation.

Two pathological κ chains which were identical in electrophoretic mobility and Inv specificity but not equivalent in re-association behaviour showed well marked differences in tryptic peptide patterns. This indicates that the number of chemically distinct human light chains exceeds the forty variants previously postulated on the basis of antigenic, allotypic and electrophoretic differences.

     

Immunogold quantitation of immunoglobulin light chains in renal amyloidosis and kappa light chain nephropathy.

By quantitative immunoelectron microscopy using protein A-gold, the authors compared the content and distribution of immunoglobulin light chain (LC) antigens in glomeruli from 11 cases of renal amyloidosis with that in two cases of kappa LC glomerulopathy and two cases of diabetic glomerulosclerosis. In a supplementary study and using a similar immunogold technique, the authors identified amyloid A in deparaffinized renal tissue from three of the 11 cases of renal amyloidosis. Each patient had similar clinical manifestations (chronic renal failure with proteinuria) and similar glomerular morphology (thickened glomerular basement membranes and nodular expansion of the mesangium). In 12 cases (10 amyloid, 2 kappa LC), immunoelectron microscopy localized LC antigens over the glomerular deposits and allowed indirect tissue quantitation of each LC antigen to the various cellular and interstitial compartments. In 6 of the 11 cases of renal amyloidosis, the amyloid labeled only for lambda, and in one, only for kappa. In one patient with Waldenström''s macroglobulinemia, who had a biclonal gammopathy, both LC were identified in the amyloid. In two cases, both of whom had a history of chronic suppurative lung disease, both LC antigens as well as amyloid A were localized to the amyloid fibrils. In only one case, in which glomerular amyloid labeled for amyloid A, the amyloid did not label for either LC. Whereas lambda LC-derived fibrils often appeared as spicules in the glomerular subepithelial space, other amyloid deposits usually accumulated in the subendothelial zone and did not form spicules. The epimembranous location of spicules suggested that the amyloid precursor protein transformed into amyloid fibrils after filtration into the urinary space. Presence of epimembranous spicules may explain the more severe proteinuric renal failure and the more rapid progression to glomerulosclerosis described in primary amyloidosis.     

Association of immunoglobulin G4 and free light chain with idiopathic pleural effusion

The cause of pleural effusion remains uncertain in approximately 15% of patients despite exhaustive evaluation. As recently described immunoglobulin (Ig)G4‐related disease is a fibroinflammatory disorder that can affect various organs, including the lungs, we investigate whether idiopathic pleural effusion includes IgG4‐associated etiology. Between 2000 and 2012, we collected 830 pleural fluid samples and reviewed 35 patients with pleural effusions undiagnosed after pleural biopsy at Yamaguchi‐Ube Medical Center. Importantly, IgG4 immunostaining revealed infiltration of IgG4‐positive plasma cells in the pleura of 12 patients (34%, IgG4⁺ group). The median effusion IgG4 level was 41 mg/dl in the IgG4⁺ group and 27 mg/dl in the IgG4^? group (P < 0·01). The light and heavy chains of effusion IgG4 antibodies of patients in the IgG4⁺ group were heterogeneous by two‐dimensional electrophoresis, indicating the absence of clonality of the IgG4 antibodies. Interestingly, the κ light chains were more heterogeneous than the λ light chains. The measurement of the κ and λ free light chain (FLC) levels in the pleural fluids showed significantly different κ FLC levels (median: 28·0 versus 9·1 mg/dl, P < 0·01) and κ/λ ratios (median: 2·0 versus 1·2, P < 0·001) between the IgG4⁺ and IgG4^? groups. Furthermore, the κ/λ ratios were correlated with the IgG4⁺/IgG⁺ plasma cell ratios in the pleura of the IgG4⁺ group. Taken together, these results demonstrate the involvement of IgG4 in certain idiopathic pleural effusions and provide insights into the diagnosis, pathogenesis and therapeutic opportunities of IgG4‐associated pleural effusion.     

Reassessment of the heavy chain deletion in human immunoglobulin Sac in the light of current theories of immunoglobulin gene assembly

IgG Sac is an immunoglobulin with large deletions in both heavy and light chains. Reexamination of the amino acid sequence of the γ chains of this immunoglobulin has led to the conclusion that the deletion in these chains, like that in the light chains, is the result of a pre-translational event, and not the result of in vivo proteolytic degradation. These chains differ from other γ heavy chain disease proteins in that the post-deletion normal sequence begins with the J segment rather than with the hinge region, and in that they are covalently associated with light chains.     

Immunohistochemical study of immunoglobulin light chain amyloidosis with antibodies to the immunoglobulin light chain variable region

To detect immunoglobulin (Ig) light chain amyloidosis (AL amyloidosis) in formalin-fixed, paraffin-embedded tissue sections by immunohistochemistry, polyclonal antibodies were generated against synthetic peptides corresponding to amino acids 1-19 of the Ig lambda light chain V lambda VI subgroup (anti-V lambda VI (1-19)) and the Ig kappa light chain Vkappa I subgroup (anti-Vkappa I (1-19)). Anti-V lambda VI (1-19) antibody reacted with amyloid deposits in 21 of 22 Alambda amyloidosis cases, and anti-Vkappa I (1-19) antibody reacted with amyloid deposits in 10 of 11 Akappa amyloidosis cases. Immunoreactivity varied in intensity by case and within specimens. Surprisingly, amyloid deposits were positive for anti-V kappa I (1-19) staining in one case of Alambda amyloidosis. Analysis of anti-V lambda VI (1-19) and anti-Vkappa I (1-19) antibody reactivity by ELISA showed some cross-reactivity with peptides other than antigen peptides. The antibodies were not reactive in all cases of AL amyloidosis examined but may be useful, together with anti-Ig constant region antibodies, for immunohistochemical diagnosis of AL amyloidosis.     

Protein-protein interactions between native Ro52 and immunoglobulin G heavy chain.

Using a yeast two-hybrid system to search for proteins interacting with Ro52 autoantigen, we identified a novel protein-protein interaction. Two different cDNA clones, which interacted with Ro52 in the yeast two-hybrid system, were identified and isolated from a human B-cell library. Surprisingly, both clones encoded the heavy chain of human IgG1. The expression of both HIS3 and beta-galactosidase reporter genes in yeast suggested that the interaction between Ro52 and IgG occurred in vivo. In vitro studies utilizing recombinant Ro52 and purified immunoglobulins indicated that the interaction was immunoglobulin class and subclass specific. Ro52 interacted with IgG1 and IgG4, but not with IgG2, IgG3, IgA or IgM. Ro52 could also precipitate IgG directly from serum. The identified cDNA clones did not include the variable region of IgG, which suggested a non-classical interaction independent of antibody specificity. We further mapped the domain of Ro52 responsible for this interaction to the C-terminus rfp-like region. In conclusion, our data support an unusual interaction between native Ro52 and IgG. The potential biological significance of this unusual protein-protein interaction is discussed.     

Synthesis of abnormal heavy and light chains in multiple myeloma with visceral deposition of monoclonal immunoglobulin.

In a patient treated for IgA kappa myeloma, bone marrow relapse and a sharp drop in the serum IgA level paralleled tissue deposition of non-amyloid material reactive with anti-kappa anti-alpha sera in immunofluorescence studies of kidney and liver biopsies. Clinical manifestations were progressive renal failure with nephrotic syndrome, with both tubular and glomerular lesions (including nodular glomerulosclerosis), hepatomegaly, cardiac and neurological symptoms. Biosynthesis experiments showed the production of alpha chains diminished in length by about one domain which were rapidly degraded predominantly after secretion and of two species of light chains; normal-sized light chains which assembled with alpha chains and abnormally short ones which were secreted as free light chains. The apparent molecular weight of the light chains was larger in secretions than in cytoplasmic extracts, suggesting their glycosylation. These results suggest a causal relationship between tissue deposition and production of abnormal immunoglobulins by a variant clone, the emergence of which was possibly induced by Melphalan therapy.     

Surface immunoglobulin receptors of rabbit lymphoid cells. Evaluation of fluorescent staining with antibodies to immunoglobulin light chain allotypes

Rabbit lymphoid cells were stained with fluorescein-labelled antiallotype antibodies. The double layer technique was found to be more sensitive than the direct staining. Rabbit B cells are stained only via their surface immunoglobulin (sIg) receptors and not via the receptors for the Fc portion of the IgG. Peritoneal exudate macrophages do not carry sIg receptors and are stained via their Fc receptors. Removal of protein aggregates from the system and use of reagents prepared from F(ab')2 immunoglobulin fragments prevent staining of the macrophages through their Fc receptors. There was a good agreement between the percent of RABELA-positive and sIg-containing spleen cells. In appendix there were approximately 20% more RABELA-positive than sIg-positive cells.     

Human myosin heavy chain genes assigned to chromosome 17 using a human cDNA clone as probe

A cDNA clone complementary to the mRNA encoding human myosin heavy chain has been isolated from a human fetal skeletal muscle cDNA library. A 600 base pair fragment of the inserted human cDNA has been used as probe in the Southern analysis of DNA from panels of rat/human and mouse/human somatic cell hybrids. All the sequences detected by this probe have been mapped to chromosome 17 in the region 17pter → 17p11. There was no evidence for MHC sequences on any other chromosome.     

Powered by pairing: the surrogate light chain amplifies immunoglobulin heavy chain signaling and pre-selects the antibody repertoire

Selective expansion of functional pre-B cells is accomplished by the assembly of a signaling-competent pre-B cell receptor (pre-BCR) consisting of immunoglobulin mu heavy chains (muHC), surrogate light chains (SLC) and Igalpha/Igbeta. Here, we review recent data showing that muHCs, in the absence of SLC, deliver autonomous differentiation signals. However, enhanced signaling necessary for pre-B cell expansion requires cross-linking of pre-BCRs via the non-immunoglobulin tail of SLC's subunit lambda5. We also discuss how SLC's ability to modulate the strength of pre-BCR signals is controlled by a muHC's idiotype and its affinity to the chaperone BiP. In this model, BiP in concert with SLC functions as a pre-selector of the antibody repertoire.