Salts enhance both protein stability and amyloid formation of an immunoglobulin light chain

Amyloid fibrils are associated with sulfated glycosaminoglycans in the extracellular matrix. The presence of sulfated glycosaminoglycans is known to promote amyloid formation in vitro and in vivo, with the sulfate groups playing a role in this process. In order to understand the role that sulfate plays in amyloid formation, we have studied the effect of salts from the Hofmeister series on the protein structure, stability and amyloid formation of an amyloidogenic light chain protein, AL-12. We have been able to show for the first time a direct correlation between protein stability and amyloid formation enhancement by salts from the Hofmeister series, where SO₄²⁻ conferred the most protein stability and enhancement of amyloid formation. Our study emphasizes the importance of the effect of ions in the protein bound water properties and downplays the role of specific interactions between the protein and ions.     

Analysis of immunoglobulin light chain loci in sheep

A sheep kappa cDNA probe was isolated, characterized by sequence analysis and shown to have significant sequence identity to other kappa light chains. This probe and a sheep lambda light chain probe were used to estimate the extent of various sheep immunoglobulin light chain gene loci by Southern blot analysis of genomic DNA. The results showed that the sheep has a single hybridizing kappa constant gene and three to five kappa V segment bands. Segregation of three polymorphic bands at the lambda C locus indicated that they were products of separate C segments. Restriction fragment pattern variations were obtained using light chain probes on various sheep breeds, but no pattern or individual band was characteristic for a particular breed.     

抗鹅免疫球蛋白轻链单克隆抗体的鉴定及其应用

免疫球蛋白是机体固有免疫系统的组成部分,是机体防御的第一道防线。本研究对抗鹅免疫球蛋白轻链单克隆抗体进行了特征分析并将其应用到不同免疫试验中用以检测鹅免疫球蛋白。用此单克隆抗体制备的免疫亲和层析柱用以分离血清中的鹅免疫球蛋白;偶联辣根过氧化物酶 (Horseradish peroxidase,HRP) 后的单克隆抗体用作第二抗体来检测鹅特异性抗体。此外,该单克隆抗体可以识别和定位外周血淋巴细胞中的SIg+淋巴细胞。研究表明,该单克隆抗体可在多种条件下检测或分离鹅免疫球蛋白并作为研究鹅体液免疫的有力工具。     

Iron loading-induced aggregation and reduction of iron incorporation in heteropolymeric ferritin containing a mutant light chain that causes neurodegeneration

Hereditary ferritinopathy (HF) is a neurodegenerative disease characterized by intracellular ferritin inclusion bodies (IBs) and iron accumulation throughout the central nervous system. Ferritin IBs are composed of mutant ferritin light chain as well as wild-type light (Wt-FTL) and heavy chain (FTH1) polypeptides. In vitro studies have shown that the mutant light chain polypeptide p.Phe167SerfsX26 (Mt-FTL) forms soluble ferritin 24-mer homopolymers having a specific structural disruption that explains its functional problems of reduced ability to incorporate iron and aggregation during iron loading. However, because ferritins are usually 24-mer heteropolymers and all three polypeptides are found in IBs, we investigated the properties of Mt-FTL/FTH1 and Mt-FTL/Wt-FTL heteropolymeric ferritins. We show here the facile assembly of Mt-FTL and FTH1 subunits into soluble ferritin heteropolymers, but their ability to incorporate iron was significantly reduced relative to Wt-FTL/FTH1 heteropolymers. In addition, Mt-FTL/FTH1 heteropolymers formed aggregates during iron loading, contrasting Wt-FTL/FTH1 heteropolymers and similar to what was seen for Mt-FTL homopolymers. The resulting precipitate contained both Mt-FTL and FTH1 polypeptides as do ferritin IBs in patients with HF. The presence of Mt-FTL subunits in Mt-FTL/Wt-FTL heteropolymers also caused iron loading-induced aggregation relative to Wt-FTL homopolymers, with the precipitate containing Mt- and Wt-FTL polypeptides again paralleling HF. Our data demonstrate that co-assembly with wild-type subunits does not circumvent the functional problems caused by mutant subunits. Furthermore, the functional problems characterized here in heteropolymers that contain mutant subunits parallel those problems previously reported in homopolymers composed exclusively of mutant subunits, which strongly suggests that the structural disruption characterized previously in Mt-FTL homopolymers occurs in a similar manner and to a significant extent in both Mt-FTL/FTH1 and Mt-FTL/Wt-FTL heteropolymers.     

Analysis of the effect of electrostatic energy truncation in molecular dynamics simulations of immunoglobulin G light chain dimer

Molecular dynamics (MD) simulations of immunoglobulin G (IgG) light chain dimer using particle mesh Ewald (PME) and cutoff methods of treating electrostatic interactions were performed. The results indicate that structural parameters (RMSD, radius of gyration, solvent accessible surface) are very similar for both schemes; however, PME simulation shows increased mobility of side chains. This leads to larger fluctuations in the distance between the monomers in the dimer molecule, and, as a consequence, results in decreased number of interactions across the dimer interface. The wall clock time of the simulations was also compared. It was shown that the PME method is approximately 30% faster than the cutoff method for the system studied on a single processor.Figure Backbone order parameters for PME (red) and cutoff (green) calculations. Thick, horizontal lines show stable secondary structures     

Characterization of a variable fragment of a human immunoglobulin lambda chain obtained by peptic cleavage in urea

The digestion of a human dimeric lambda Bence Jones protein with pepsin in 8 M urea without prior reduction and alkylation produced a fragment of molecular weight 11,000, designated as upV lambda, corresponding to almost the entire variable region of an immunoglobulin light chain. The C-terminal amino acid residue of fragment upV lambda was shown to correspond to leucine at position 104 of the immunoglobulin lambda light chain. Fragment upV lambda was isolated in the yield of 30% by ion exchange chromatography after gel filtration. When another lambda Bence Jones protein was treated in the same way, similar results were obtained.     

Cyanamide mediated syntheses of peptides containing histidine and hydrophobic amino acids

Summary Using the model of a primitive earth evaporation pond, the synthesis of three histidyl peptides in yields of up to 11% was demonstrated when aqueous solutions of histidine, leucine, ATP, cyanamide, and MgCl₂ were evaporated and heated for 24 h at 80°C. In addition, peptides were formed in yields of up to 56%, 35%, and 21%, respectively for phenylalanine, leucine, and alanine when aqueous solutions of the appropriate amino acid were evaporated and heated with cyanamide and one or more of the following components: ATP, AMP, 4-amino-5-imidazole carboxamide, or MgCl₂. The greatest peptide yield occurred at pH 3. But peptide formation was demonstrated for a system of Leu, cyanamide, and MgCl₂ adjusted to pH 7 with NH₄OH.Peptide synthesis was also studied in the presence of CaCl₂, ZnCl₂, different adenosine nucleotides, and UTP to compare their effects on peptide synthesis. The optimum conditions for cyanamide mediated peptide synthesis were also studied in terms of pH, reaction time, reaction temperature, and cyanamide concentration. The major side product in nearly all reactions studied appears to be an amino acid-cyanamide adduct. Peptides were analyzed and identified by thin layer chromatography, acid hydrolysis, and enzymatic degradation.     

Myosin light chain kinase and the role of myosin light chain phosphorylation in skeletal muscle

Skeletal muscle myosin light chain kinase (skMLCK) is a dedicated Ca²⁺/calmodulin-dependent serine–threonine protein kinase that phosphorylates the regulatory light chain (RLC) of sarcomeric myosin. It is expressed from the MYLK2 gene specifically in skeletal muscle fibers with most abundance in fast contracting muscles. Biochemically, activation occurs with Ca²⁺ binding to calmodulin forming a (Ca²⁺)₄•calmodulin complex sufficient for activation with a diffusion limited, stoichiometric binding and displacement of a regulatory segment from skMLCK catalytic core. The N-terminal sequence of RLC then extends through the exposed catalytic cleft for Ser15 phosphorylation. Removal of Ca²⁺ results in the slow dissociation of calmodulin and inactivation of skMLCK. Combined biochemical properties provide unique features for the physiological responsiveness of RLC phosphorylation, including (1) rapid activation of MLCK by Ca²⁺/calmodulin, (2) limiting kinase activity so phosphorylation is slower than contraction, (3) slow MLCK inactivation after relaxation and (4) much greater kinase activity relative to myosin light chain phosphatase (MLCP). SkMLCK phosphorylation of myosin RLC modulates mechanical aspects of vertebrate skeletal muscle function. In permeabilized skeletal muscle fibers, phosphorylation-mediated alterations in myosin structure increase the rate of force-generation by myosin cross bridges to increase Ca²⁺-sensitivity of the contractile apparatus. Stimulation-induced increases in RLC phosphorylation in intact muscle produces isometric and concentric force potentiation to enhance dynamic aspects of muscle work and power in unfatigued or fatigued muscle. Moreover, RLC phosphorylation-mediated enhancements may interact with neural strategies for human skeletal muscle activation to ameliorate either central or peripheral aspects of fatigue.     

Removal of a C-terminal serine residue proximal to the inter-chain disulfide bond of a human IgG1 lambda light chain mediates enhanced antibody stability and antibody dependent cell-mediated cytotoxicity

Optimization of biophysical properties is a critical success factor for the developability of monoclonal antibodies with potential therapeutic applications. The inter-domain disulfide bond between light chain (Lc) and heavy chain (Hc) in human IgG1 lends structural support for antibody scaffold stability, optimal antigen binding, and normal Fc function. Recently, human IgG1λ has been suggested to exhibit significantly greater susceptibility to reduction of the inter Lc-Hc disulfide bond relative to the same disulfide bond in human IgG1κ. To understand the molecular basis for this observed difference in stability, the sequence and structure of human IgG1λ and human IgG1κ were compared. Based on this Lc comparison, three single mutations were made in the λ Lc proximal to the cysteine residue, which forms a disulfide bond with the Hc. We determined that deletion of S214 (dS) improved resistance of the association between Lc and Hc to thermal stress. In addition, deletion of this terminal serine from the Lc of IgG1λ provided further benefit, including an increase in stability at elevated pH, increased yield from transient transfection, and improved in vitro antibody dependent cell-mediated cytotoxicity (ADCC). These observations support the conclusion that the presence of the terminal serine of the λ Lc creates a weaker inter-chain disulfide bond between the Lc and Hc, leading to slightly reduced stability and a potential compromise in IgG1λ function. Our data from a human IgG1λ provide a basis for further investigation of the effects of deleting terminal serine from λLc on the stability and function of other human IgG1λ antibodies.     

Cell cycle kinetics of the accumulation of heavy and light chain immunoglobulin proteins in a mouse hybridoma cell line

Rates of accumulation of immunoglobulin proteins have been determined using flow cytometry and population balance equations for exponentially growing murine hybridoma cells in the individual G₁, S and G₂+M cell cycle phases. A producer cell line that secretes monoclonal antibodies, and a nonproducer clone that synthesizes only -light chains were analyzed. The pattern for the kinetics of total intracellular antibody accumulation during the cell cycle is very similar to the previously described pattern for total protein accumulation (Kromenaker & Srienc 1991). The relative mean rate of heavy chain accumulation during the S phase was approximately half the relative mean rate of light chain accumulation during this cell cycle phase. This indicates an unbalanced synthesis of heavy and light chains that becomes most pronounced during this cell cycle phase. The nonproducer cells have on average an intracellular light chain content that is 42% lower than that of the producer cells. The nonproducer cells in the G₁ phase with low light chain content did not have a significantly higher rate of light chain accumulation relative to other G₁ phase nonproducer cells. This is in sharp contrast to what was observed for the G₁ phase producer cells. In addition, although the relative mean rate of accumulation of light chain was negative for G₂+M phase nonproducer cells, the magnitude of this relative mean rate was less than half that observed for the producer cells in this cell cycle phase. This suggests that the mechanisms that regulate the transport of fully assembled antibody molecules through the secretion pathway differ from those which regulate the secretion of free light chains. The results reported here indicate that there is a distinct pattern for the cell cycle dynamics of antibody synthesis and secretion in hybridomas. These results are consistent with a model for the dynamics of secretion which suggests that the rate of accumulation of secreted proteins will be greatest for newborn cells due to an interruption of the secretion pathway during mitosis.     

The reported germline repertoire of human immunoglobulin kappa chain genes is relatively complete and accurate

We describe a bioinformatic analysis of germline and rearranged immunoglobulin kappa chain (IGK) gene sequences, performed in order to assess the completeness and reliability of the reported IGK repertoire. In contrast to the reported heavy-chain gene repertoire, which includes many dubious sequences, only five IGK variable gene (IGKV) alleles appear to have been reported in error. There was, however, insufficient evidence to justify removing these IGKV genes from the germline repertoire. Bioinformatic analysis of apparent mismatches between reported germline genes and 1,863 expressed IGK sequences suggested the existence of two unreported IGKV polymorphisms. Genomic screening of 12 individuals led to the confirmation of both of these polymorphisms, IGKV1-16*02 and IGKV2-30*02. We also show that in contrast to the heavy chain, the IGK repertoire is dominated by sequences that use just a handful of kappa variable (IGKV) and junction (IGKJ) gene pairs. There is also little modification of IGKV and IGKJ genes by the processes of exonuclease removal and N nucleotide addition. The expressed IGK repertoire therefore lacks diversity and the junction region is particularly constrained. Remarkably, the analysis of a dataset of 435 relatively unmutated rearranged kappa genes showed that ten amino acid sequences account for almost 10% of the rearrangements, with identical sequences being derived from as many as seven independent sources. Such dominant sequences are likely to have important roles in the operation of the humoral immune response.     

Improvement of antigen binding ability of human antibodies by light chain shifting

Human HB4C5 hybridoma cells produce a lung cancer-specific IgM human monoclonal antibody (mAb). HB4C5 human mAb cross-reacts with Candida cytochrome c (Cyt c) and carboxypeptidase (Cpase). Concanavalin A (ConA)-resistant variants of HB4C5 cells loss the original light chain followed by expression of various new light chains at a high incidence (light chain shifting) (Tachibana et al., 1996). HTD8 cells, one of the ConA-resistant variant subclones of HB4C5 cells, undergo the active light chain shifting and produce various sublines, each of which stably secretes new mAb consisting of a new light chain and a HB4C5 heavy chain. The new mAb exhibits altered antigen binding ability from that of the original antibody. We could expect that HTD8 cells can be used as ‘a light chain stem cell line’ to improve antigen binding ability and specificity of established human mAbs. A BD9D12 IgG human mAb recognizes lung cancer cells and cross-reacts with cytokeratin 8. Introduction of the heavy chain gene of BD9D12 mAb into HTD8 cells resulted in establishment of various sublines which secreted various kinds of hybrid antibodies consisting of different light chains derived from HTD8 subclones which underwent light chain shifting and a common IgG heavy chain derived from BD9D12. These hybrid antibodies exhibited different or improved reactivities to Cyt, Cpase, cytokeratin 8 and various cancer cells from those of parental mAb, demonstrating that light chain shifting can be applied to improve the affinity and specificity of human mAb. This revised version was published online in June 2006 with corrections to the Cover Date.     

A fluorescent resonant energy transfer-based biosensor reveals transient and regional myosin light chain kinase activation in lamella and cleavage furrows

Approaches with high spatial and temporal resolution are required to understand the regulation of nonmuscle myosin II in vivo. Using fluorescence resonance energy transfer we have produced a novel biosensor allowing simultaneous determination of myosin light chain kinase (MLCK) localization and its [Ca2+]4/calmodulin-binding state in living cells. We observe transient recruitment of diffuse MLCK to stress fibers and its in situ activation before contraction. MLCK is highly active in the lamella of migrating cells, but not at the retracting tail. This unexpected result highlights a potential role for MLCK-mediated myosin contractility in the lamella as a driving force for migration. During cytokinesis, MLCK was enriched at the spindle equator during late metaphase, and was maximally activated just before cleavage furrow constriction. As furrow contraction was completed, active MLCK was redistributed to the poles of the daughter cells. These results show MLCK is a myosin regulator in the lamella and contractile ring, and pinpoints sites where myosin function may be mediated by other kinases.     

Allelic variants of rearranged immunoglobulin heavy and light chain genes in hybridoma PTF-02 and parent myeloma

The hybridoma PTF-02 secretes an antibody against pig transferrin. Rearranged genes for heavy and light immunoglobulin chains have been studied in the genomes of this hybridoma and in the parent myeloma P3-X63.Ag8.653. The hybridoma was shown to contain three rearranged allelic variants of the heavy chain gene's locus. The gene H2, responsible for synthesis of the heavy chain of the antibody to transferrin, was transmitted in the hybridoma cell from a lymphocyte. Two other genes (H1 and H3) were found both in the hybridoma and parent myeloma genomes. The gene H1 was identified in MOPC21 myeloma, which is a precursor of the X63.Ag8 descendent line. Rearranged k genes were also identified both in the hybridoma and parent myeloma. A functional (K2) gene and a fetal (F) gene appeared in the hybridoma genome from an antigen-stimulated normal lymphocyte. The fetal gene was lost in the course of continuous cultivation of the hybridoma PTF-02 cell line. The gene K1 was transmitted from the myeloma used for fusion. In such a way, the pedigree of rearranged heavy and light chain genes in the hybridoma PTF-02 was established. The results obtained in this work may be relevant to many hybridomas whose immortalizing fusion partner is a MOPC21 derivative, and allow one to identify and isolate functional variable genes to create recombinant constructions.     

Regulation of LPA-promoted myofibroblast contraction: role of Rho, myosin light chain kinase, and myosin light chain phosphatase

Myofibroblasts generate the contractile force responsible for wound healing and pathological tissue contracture. In this paper the stress-relaxed collagen lattice model was used to study lysophosphatidic acid (LPA)-promoted myofibroblast contraction and the role of the small GTPase Rho and its downstream targets Rho kinase and myosin light chain phosphatase (MLCPPase) in regulating myofibroblast contraction. In addition, the regulation of myofibroblast contraction was compared with that of smooth muscle cells. LPA-promoted myofibroblast contraction was inhibited by the myosin light chain kinase (MLCK) inhibitors KT5926 and ML-7; however, in contrast to that observed in smooth muscle cells, elevation of intracellular calcium alone was not sufficient to promote myofibroblast contraction. These results suggest that Ca(2+)-mediated activation of MLCK, while necessary, is not sufficient to promote myofibroblast contraction. The specific Rho inactivator C3-transferase and the Rho kinase inhibitor Y-27632 inhibited LPA-promoted myofibroblast contraction, suggesting that contraction depends on activation of the Rho/Rho kinase pathway. Calyculin, a type 1 phosphatase inhibitor known to inhibit MLCPPase, could promote myofibroblast contraction in the absence of LPA, as well as restore contraction in the presence of C3-transferase or Y-27632. Together these results support a model whereby Rho/Rho kinase-mediated inhibition of MLCPPase is necessary for LPA-promoted myofibroblast contraction, in contrast to smooth muscle cells in which Ca(2+) activation of MLCK alone is sufficient to promote contraction.