Amino terminal sequence of heavy and light chains from ratfish immunoglobulin

The ratfish,Callorhinchus callorhinchus, a representative of the Holocephali, has a natural serum hemagglutinin (M _r 960 000), composed of heavy (M _r 71000), light (M _r 22 500), and J (M _r 16 000) chains. To approach the mechanisms that generate diversity at this level of evolution, the amino terminal sequence of the heavy and light chains was determined by automated microsequencing. The chains are unblocked and have modest internal sequence heterogeneity. The heavy chains show sequence similarity with the terminal region of the heavy chain from the horned shark,Heterodontus francisci, and other species. In contrast to the heavy chain, the ratfish light chains display low sequence similarity with their shark kappa counterparts. However, their similarity with the variable region of the chicken lambda light chains is about 75%.     

Co-translational modification of nascent immunoglobulin heavy and light chains

We have investigated the in vivo co-translational covalent modification of nascent immunoglobulin heavy and light chains. Nascent polypeptides were separated from completed polypeptides by ion-exchange chromatography of solubilized ribosomes on QAE-Sephadex. First, we have demonstrated that MPC 11 nascent heavy chains are quantitatively glycosylated very soon after the asparaginyl acceptor site passes through the membrane into the cisterna of the rough endoplasmic reticulum. Nonglycosylated completed heavy chains of various classes cannot be glycosylated after release from the ribosome, due either to rapid intramolecular folding and/or intermolecular assembly, which cause the acceptor site to become unavailable for the glycosylation enzyme. Second, we have shown that the formation of the correct intrachain disulfide loop within the first light chain domain occurs rapidly and quantitatively as soon as the appropriate cysteine residues of the nascent light chain pass through the membrane into the cisterna of the endoplasmic reticulum. The intrachain disulfide loop in the second or constant region domain of the light chain is not formed on nascent chains, because one of the cysteine residues involved in this disulfide bond does not pass through the endoplasmic reticulum membrane prior to chain completion and release from the ribosome. Third, we have demonstrated that some of the initial covalent assembly (formation of interchain disulfide bonds) occurs on nascent heavy chains prior to their release from the ribosome. The results are consistent with the pathway of covalent assembly of the cell line, in that completed light chains are assembled onto nascent heavy chains in MPC 11 cells (IgG₂b), where a heavy-light half molecule is the major initial covalent intermediate; and completed heavy chains are assembled onto nascent heavy chains in MOPC 21 cells (IgG₁), where a heavy chain dimer is the major initial disulfide linked intermediate.     

Cytotoxicity of amyloidogenic immunoglobulin light chains in cell culture

Light-chain amyloidosis (AL) is a devastating protein-misfolding disease characterized by abnormal proliferation of plasma cells in the bone marrow that secrete monoclonal immunoglobulin light chains that misfold and form amyloid fibrils, thus causing organ failure and death. Numerous reports on different protein-misfolding diseases show that soluble oligomeric species populated by amyloidogenic proteins can be quite toxic to cells. However, it is not well established whether the soluble immunoglobulin light-chain species found in circulation in patients with AL are toxic to cells in target organs. We determined the cellular toxicity of two well-characterized light-chain variable domain proteins from cardiac AL patients and their corresponding germline protein, devoid of somatic mutations. Our results show that the soluble form of the AL proteins we characterized are toxic to cardiomyocytes, and that the species found in cell culture correspond, for the most part, to the species present in circulation in these patients.     

Free immunoglobulin light chains: A role in minimal change disease

Immunoglobulins are tetrameric molecules consisting of two heavy and two light chains linked by disulfide bonds. Single light chains are normally secreted in the plasma under soluble form. These immunoglobulin free light chains circulating in the blood may hold unexpected roles in diseases.Minimal change disease is defined as a renal disease with massive proteinuria and no obvious damage on light microscopy. We hypothesize that minimal change disease is not a primary renal disease but an immune disease due to a defect in B cells mediated by a special immunoglobulin chain. The efficiency of drugs targeting the immune system and the association to Hodgkin disease as well as: (1) the efficiency of B cell depletion to prevent relapse; (2) the association with B leukemia; and (3) the activation of CD23 during relapse point up a primary involvement of B cells. We hypothesize that an immunoglobulin chain with special polymorphism might be the link between the immune system and the dysfunction of the glomerular wall while immunoglobulin depletion leads to a transient remission of proteinuria in graft recurrence of the disease and nephropathy mediated by a monoclonal immunoglobulin chain may feature minimal change disease.Other diseases where free light chains may be involved include atopy, thromboembolism, glomerular inflammatory diseases and autoimmune diseases. We conclude that free circulating light chains, through infinite possibilities of polymorphisms determined by the variable domain are potential disturbing agents of many biological cascades or structures and could likely play the first role in multiple diseases.     

An allotypic marker on chicken immunoglobulin light chains.

The first chicken immunoglobulin light (L) chain allotypic specificity (L-1.1) to be described that was present on IgM, 7S Ig, Fab, and L chains was detected by radioimmunoassay. The gene controlling the expression of L-1.1 is inherited in a simple Mendelian fashion at an autosomal locus and is unlinked to a constant region heavy chain locus, four blood group loci and three loci determining lymphocyte cell surface alloantigens.     

Assembly and plasma membrane targeting of recombinant immunoglobulin chains in plants with a murine immunoglobulin transmembrane sequence

The cDNA encoding a full-length murine immunoglobulin 1 heavy chain with its native leader sequence, transmembrane and intracellular domains was introduced into transgenic plants. Transformed plants expressed the recombinant polypeptide, but, in contrast to plants expressing the heavy chain without transmembrane sequence, the protein appeared to be associated with a plant cell membrane. Extraction of the membrane-associated heavy chain required the presence of a non-ionic detergent, and immunofluorescence studies of protoplasts demonstrated surface expression of membrane Ig heavy chain on up to 40% of the cells from a transgenic leaf. In plants expressing both the membrane Ig heavy chain and its partner light chain, functional antibody was also localised to the plant cell membrane and retention of the heavy chain at this site appeared to have no effect on the efficiency of antibody assembly. This approach of localising and accumulating recombinant antibody in cell membranes may have a number of applications, including passive immunisation against plant pathogens.     

Localization of a conformational epitope common to non-native and fibrillar immunoglobulin light chains

Amyloid fibrils and partially unfolded intermediates may be distinguished serologically from native amyloidogenic precursor proteins or peptides. In this regard, we had previously reported that the IgG1 mAb 11-1F4, generated by immunizing mice with a thermally denatured variable region fragment of the human Igkappa4 Bence Jones protein Len, reacted specifically with light chain (LC) fibrils, irrespective of kappa or lambda isotype but, notably, did not with native molecules (Hrncic, R. et al. (2000) Am. J. Pathol. 157, 1239-1246). To elucidate the molecular basis of this specificity, we have used a europium-linked fluorescent immunoassay, where it was demonstrated through epitope mapping that mAb 11-1F4 recognizes a conformational determinant contained within the first (N-terminal) 18 amino acids of misfolded LCs. The nature of this epitope was evidenced in competition studies where the peptide Len (1-18), but not the intact protein or other LCs, inhibited the binding of the antibody to fibrils. This unique reactivity was dependent on the structural integrity of this portion of the molecule, particularly the presence of a highly conserved prolyl residue at position 8. On the basis of our experimental data, we posit that the mAb 11-1F4 binding site found on partially denatured and fibrillar LCs involves an inducible N-terminal main chain reversal that results in the formation of a proline anchored beta-turn. Our delineation of this LC fibril-associated epitope provides the rationale for the design of novel amyloid-reactive antibodies with diagnostic and therapeutic potential for patients with LC-associated and other forms of amyloidosis.     

Glycosaminoglycans promote fibril formation by amyloidogenic immunoglobulin light chains through a transient interaction

Amyloid formation occurs when a precursor protein misfolds and aggregates, forming a fibril nucleus that serves as a template for fibril growth. Glycosaminoglycans are highly charged polymers known to associate with tissue amyloid deposits that have been shown to accelerate amyloidogenesis in vitro. We studied two immunoglobulin light chain variable domains from light chain amyloidosis patients with 90% sequence identity, analyzing their fibril formation kinetics and binding properties with different glycosaminoglycan molecules. We find that the less amyloidogenic of the proteins shows a weak dependence on glycosaminoglycan size and charge, while the more amyloidogenic protein responds only minimally to changes in the glycosaminoglycan. These glycosaminoglycan effects on fibril formation do not depend on a stable interaction between the two species but still show characteristic traits of an interaction-dependent mechanism. We propose that transient, predominantly electrostatic interactions between glycosaminoglycans and the precursor proteins mediate the acceleration of fibril formation in vitro.     

Plural light chains in a single plasma cell of a monoclonal gammopathy undetermined significance case: An ultrastructural study

A 44-year-old man was found to have M-proteins of IgG consisting of kappa- and lambda-chains in serum without lymphadenopathy or splenomegaly. The serum concentrations of IgG, IgA and IgM were within normal limits. Bone marrow examination showed normal cellular marrow containing 6.3% of plasma cells with no abnormal features. No chromosomal abnormality was observed at all. The patient was diagnosed as having monoclonal gammopathy of undetermined significance. The bone marrow plasma cells possessed free kappa- and lambda-chains in Golgi apparatus, rough endoplasmic reticula and cytoplasmic matrices. Plural light chains were simultaneously produced with the same heavy chain in a plasma cell by immunoelectron microscopy. This is the first report in the world of a monoclonal gammopathy of undetermined significance producing plural light chains with the same heavy chain.     

Species specificity in the isoelectric spectra of immunoglobulin light chains.

Light chains isolated from normal immunoglobulin give rise to a finite number of discrete bands when analyzed by isoelectric focusing in polyacrylamide gel. The positions and relative intensities of the bands were identical in light chains isolated from different individuals of a species, but clear differences were evident when light chains of several mammalian species were compared.     

Site-specific photobiotinylation of antibodies, light chains, and immunoglobulin fragments

The high affinity of biotin for avidin has been exploited for many antibody-based assays. This requires that biotin is covalently conjugated to the antibody molecule. Several chemically reactive biotinylation reagents are commercially available. Except for the attachment via sulfhydryl groups in the immunoglobulin (Ig) molecule, these reagents attach biotin randomly to various amino acid side chains. Although non-site-specific modification of antibodies does not interfere in most immunoassays, specific application and sensitive antibodies would benefit from site-specific biotinylation. Here we describe an affinity biotinylation technique based on a photoreactive biotin reagent. The design of this reaction was possible from the discovery of a conserved binding site in the variable Ig domain for nucleotides and nucleosides. The described photoaffinity biotinylation offers the advantages of ease, convenience, and production of a reproducible and defined biotinylated antibody preparation.     

Characterization of protein aggregation: the case of a therapeutic immunoglobulin

In this paper, a therapeutic immunoglobulin (Antibody A) has been characterized in two solutions: (1) 0.1% acetic acid containing 50 mM magnesium chloride, a solution in which the immunoglobulin is stable, and (2) 10 mM sodium phosphate buffer pH approximately 7. The protein solutions were characterized by microscopy, asymmetrical flow field-flow fractionation (FFF), light scattering, circular dichroism, fluorescence and fluorescence lifetime spectroscopy. The results show that Antibody A dissolved in 0.1% acetic acid containing 50 mM magnesium chloride exists as 88% monomer, 2% low molecular weight aggregates and 10% high molecular weight aggregates (>1 million Dalton). In phosphate buffer, Antibody A formed micrometre-sized aggregates that were best characterized by fluorescence microscopy. The aggregation of Antibody A in phosphate buffer was shown to be concomitant with conformational changes in amino acid residue side chains. The aggregates formed in phosphate buffer were easily disrupted during FFF analysis, indicating that they are formed by weak interactions. The combination of microscopy, asymmetrical flow field-flow fractionation (FFF) and spectroscopy allowed a reliable assessment of protein self association and aggregation.     

Development of quantitative immunoenzyme and radioimmunologic analysis of lambda-free immunoglobulin light chains

New quantitative techniques of radioimmunoassay (RIA) and enzyme immunoassay (EIA) were developed for determination of free light lambda-chains of immunoglobulins (immunometric sandwich-like variants) in biological fluids using two types of monoclonal specific antibodies (MAB-1 and MAB-2) to different epitopes of the antigen molecule: MAB-1 were immobilized on a solid phase (polystyrene beads), whereas MAB-2 were labeled with iodine or with the enzyme. The test systems prepared can be used for determination of concentrations from 25 to 1000 ng/ml, are very sensitive (25 ng/ml), and the analysis time is 5 h. The two methods were compared, and their clinical and diagnostic validity was evaluated in patients with various diseases associated with disorders in the antibody synthesis by the immune system.