Molecular Weight Light Chain Research Articles

Detection of High Molecular Weight Light Chain Oligomers in Urinary Exosomes of Patients with AL Amyloidosis.

Abstract 4886Detection of high molecular weight light chain oligomers in urinary exosomes of patients with AL amyloidosis. BackgroundExosomes are microvesicles that are part of the multivesicular body (MVB) pathway. They are created by the inward budding of the cell surface membrane and contain both surface bound membrane proteins and cytosolic proteins which can be used to identify the cell of origin. Immunoglobulin light chain amyloidosis (AL) occurs as the result of amyloid formation by the misfolding of monoclonal light chains (LC) and deposition of these amyloid fibrils in various soft tissues. This reaction requires the organization of the monoclonal LC's into protofibrils which are then weave into amyloid fibrils. This study was undertaken to determine whether urinary exosomes of glomerular origin contain intermediate species of amyloid formation. MethodUrine samples from patients with AL, light chain deposition disease (LCDD), multiple myeloma (MM) and monoclonal clonal gammopathy of undetermined significance (MGUS) were collected. Urinary exosomes were isolated and separated into fractions by gradient centrifugation. Western blots were performed on the urinary exosome fractions using anti-kappa or anti-lambda antibodies. Glomerular fractions were identified using antibodies directed toward podocin. ResultsUrine samples were collected from 5 patients with AL, 2 from LCDD, 1 from MM and 1 MGUS. On the Western blot, immunoglobulin LC were seen in all exosomal fractions in patients with AL amyloidosis, LCDD, MM but not MGUS which is similar to normal controls (not shown). In patients with AL, oligomeric species were found in the highest concentrations in fraction 4 and 5 (Figure 1). Fraction 4 and 5 were also stained for podocin, a glomerular protein (not shown). The highest molecular weight species was ∼250 kd which corresponds to a LC decamer. High molecular weight species were also identified in 1 of 2 LCDD patients corresponding to a tetramer. The band was identified in fraction 10 which had polycystin-1 expression suggesting a tubular origin. No high molecular weight LC species was found in patients with MM or MGUS. ConclusionOur study found high molecular weight LC species corresponding to the intermediates involved in protofibril formation in urinary exosomes of patients with AL. Smaller (tetramer) high molecular weight LC species were also found in a patient with LCDD but not in patients with MM and MGUS. Not only were the high molecular weight LC species found exclusively in the diseases characterized by deposition of LC aggregates, they were also found in the segments of the nephron where the deposits were expected: glomerulus for AL and tubular epithelium for LCDD. This is consistent with our current understanding of the pathogenic mechanisms of these diseases. We believe urinary exosomes are a powerful tool in the study of diseases involving self-aggregation of monoclonal proteins. It has tremendous potential in both diagnostic and scientific research in this area. [Display omitted] DisclosuresGertz:celgene: Honoraria; millenium: Honoraria, Membership on an entity's Board of Directors or advisory committees.

Structure of Tctex-1 and Its Interaction with Cytoplasmic Dynein Intermediate Chain

The minus-ended microtubule motor cytoplasmic dynein contains a number of low molecular weight light chains including the 14-kDa Tctex-1. The assembly of Tctex-1 in the dynein complex and its function are largely unknown. Using partially deuterated, (15)N,(13)C-labeled protein samples and transverse relaxation-optimized NMR spectroscopic techniques, the secondary structure and overall topology of Tctex-1 were determined based on the backbone nuclear Overhauser effect pattern and the chemical shift values of the protein. The data showed that Tctex-1 adopts a structure remarkably similar to that of the 8-kDa light chain of the motor complex (DLC8), although the two light chains share no amino acid sequence homology. We further demonstrated that Tctex-1 binds directly to the intermediate chain (DIC) of dynein. The Tctex-1 binding site on DIC was mapped to a 19-residue fragment immediately following the second alternative splicing site of DIC. Titration of Tctex-1 with a peptide derived from DIC, which contains a consensus sequence R/KR/KXXR/K found in various Tctex-1 target proteins, indicated that Tctex-1 binds to its targets in a manner similar to that of DLC8. The experimental results presented in this study suggest that Tctex-1 is likely to be a specific cargo adaptor for the dynein motor complex.

Cloning and expression of human V-genes derived from phage display libraries as fully assembled human anti-TNFα monoclonal antibodies

Background: With the advent of phage antibody libraries, access to completely human antibody fragments is feasible, either by direct selection from human antibody libraries, or by guided selection. After selection, Fabs and scFvs may need to be expressed as complete antibodies in mammalian cells for further characterisation, or if effector functions are required. Objectives: To rebuild and express the human anti-TNFα antibody Fab-P3A2 (isolated as a Fab fragment from phage display libraries by guided selection) as a fully assembled, functional human antibody (y-1, λ) in Sp2/0 myeloma cells, and to perform preliminary characterisation studies of the secreted IgGI molecule. A further objective was to investigate the kinetics of human antibody production and the stability of antibody secretion in transfectomas cultured in various media formulations. Study design: A tripartite strategy was employed for cloning heavy chain gene (VH)-P3 and light chain gene Vλ-A2-Cλ into mammalian cell expression vectors pαLys-30 and pαLys-17 respectively. The cell line P3A2.B5 was isolated after co-transfection of Sp2/0 mouse myelomas with the constructs, expanded and weaned into a protein free medium. Fully assembled Ig-P3A2 antibody was purified by Protein A affinity chromatography and characterised with respect to size of antibody chains, and affinity for human TNFα. Stability of secretion was investigated by extended serial sub-culture and analysis of P3A2.B5 sub-clones. Strategies of media enrichment were tested for any effect on antibody productivity by selected MAIM sub-clones. Results: The cell line P3A2.B5 secreted an assembled, human antibody Ig-P3A2, with heavy and light chains of molecular weight 55 and 28 KD respectively. Equilibrium capture studies showed Ig-P3A2 to have a dissociation constant of approximately 1.5 × 10−8 M. The mean specific productivity of the cell line increased from 1.2 pg/cell/day to 7.8 pg/cell/day by a combination of medium enrichment and serum reduction. Prolonged serial sub-culture of P3A2.B5 showed the cell line to be unstable with respect to antibody secretion. Conclusion: We have outlined a method for expression of human V genes as assembled antibodies in Sp2/0 myeloma cells. A cloning strategy for the stable expression of scFv or Fab genes isolated from phage display libraries as assembled human antibodies of the IgG1 subclass in Sp2/0 myeloma cells has been described. For maximising specific productivity of antibody-producing cell lines, supplementation of culture media with glucose, glutamine and amino acids increases antibody yield significantly compared to that in conventional media, indicating the latter is stoichiometrically limiting for production purposes.

Why aren't embryos immunologically rejected by their mothers?

Just over 50 years ago, Peter Gorer discovered the major histocompatibility complex (MHC) of the mouse, the H-2 complex (Gorer, 1936a,b; 1937). Since that time, an MHC has been defined for most mammals, and for a variety of lower vertebrate organisms (reviewed in Klein, 1986). The MHC is a set of genes that encodes the information for proteins found on the surface of cells of each individual. The proteins are highly polymorphic, and each individual in the species has a particular set of proteins defining that individual’s tissue type or MHC phenotype. The products of the MHC genes are crucial for the control of a variety of biological functions, including cell-cell interactions in the immune response, tumor and graft rejection, and reproductive success. The best-studied MHC is that of the mouse. A diagram of the current view of the mouse MHC in shown in Figure 1. Extensive work on cloning and sequencing the genes of the H-2 complex, initiated in 1980, has shown that the H-2 complex encodes at least 50 genes (Steinmetz, 1987). The MHC genes code for three different types of proteins, designated Class I, Class II, and Class III. Class I molecules are membrane-bound glycoproteins, all of which have a similar structure, a 35-45,000 molecular weight heavy chain and a 12,000 molecular weight light chain. The heavy chain is encoded in the H-2 complex, but the light chain, 132 -microglobulin, is encoded on mouse chromosome 2. In most species, the Class I genes are clustered at the telomeric end of the MHC, but in the mouse, two Class I genes have been translocated to the centromeric end of the MHC gene complex.

Structure of Myosin Subfragment-1 from Electron Microscopy and Crystallography

The primary role of the interaction of actin and myosin is the generation of force and motion as a direct consequence of the cyclic interaction of myosin crossbridges with actin filaments. Myosin is composed of six polypeptides: two heavy chains of molecular weight 220,000 daltons and two pairs of light chains of molecular weight 17,000-23,000. The C-terminal portions of the myosin heavy chains associate to form an α-helical coiled-coil rod which is responsible for myosin filament formation. The N-terminal portion of each heavy chain associates with two different light chains to form a globular head that binds actin and hydrolyses ATP. Myosin can be fragmented by limited proteolysis into several structural and functional domains. It has recently been demonstrated using an in vitro movement assay that the globular head domain, subfragment-1, is sufficient to cause sliding movement of actin filaments.The discovery of conditions for crystallization of the myosin subfragment-1 (S1) has led to a systematic analysis of S1 structure by x-ray crystallography and electron microscopy. Image analysis of electron micrographs of thin sections of small S1 crystals has been used to determine the structure of S1 in the crystal lattice.

Structural analysis of myosin genes using recombinant DNA techniques.

Myosin, the major protein of the myofibril, consists of two heavy chains with a molecular weight (MW) of 200,000, complexed with four light chains of MW 17,000 to 21,000. Both the heavy and light chains exhibit polymorphisms that are tissue-specific and developmental stage-specific. Myosin heavy chains and light chains appear to be represented in the genome as multigene families in various species, including chickens, cattle, humans, rats, rabbits and nematodes. Myosin heavy-chain proteins have a high amino acid sequence homology among isoforms, and the genes for each isoform likewise exhibit a high degree of nucleotide sequence conservation. The myosin heavy-chain genes have a complex structure and contain up to 65% intervening sequence composed of up to 20 or more individual introns. Partial sequence data, transcriptional orientation and tissue of expression have been determined for several myosin heavy-chain genes. The use of recombinant DNA and associated techniques will eventually yield definitive information on the control of expression of each individual gene, as well as factors that regulate expression of closely related isoforms.

Immunoglobulin classes in the garden lizard, [formula omitted][formula omitted

Two classes of immunoglobulins have been purified from lizard serum using a combination of ion-exchange chromatography on DEAE-cellulose and gel filtration on Sephadex G-200 or on Sepharose 6B. Lizard IgM is 2-ME sensitive and has an intact molecular weight similar to human IgM. On SDS-PAGE, reduced IgM dissociates into heavy and light chains of molecular weight 70,000 and 23,500 daltons respectively. Lizards also possess a 2-ME resistant, low molecular weight immunoglobulin designated as IgY similar to avian and amphibian IgY. IgY dissociates on SDS-PAGE to yield 59,500 dalton heavy and 26,000 dalton light chains. Antisera raised in rabbits to each of the two Ig classes could be made class-specific by cross-absorption, thus indicating that IgM and IgY represent distinct isotypes.

Ordered phosphorylation of the two 20 000 molecular weight light chains of smooth muscle myosin.

The time courses of phosphorylation of the Mr 20 000 light chains by purified myosin light chain kinase plus calmodulin were determined. In confirmation of an earlier report [Persechini, A., & Hartshorne, D. J. (1981) Science (Washington, D.C.) 213, 1383-1385], a steady-state kinetic analysis indicates that the phosphorylation occurs in an ordered manner; i.e., at a phosphorylation level of 0.5 mol of 32P incorporated per mol of bound Mr 20 000 light chain, each myosin molecule would have one phosphorylated head. The kinetic parameters obtained for the phosphorylation of the more reactive myosin head are similar to those determined by using isolated light chains. It is suggested that the ordered, or sequential, phosphorylation, and the different reactivities of the two Mr 20 000 light chains, is the result of preexisting asymmetry of the myosin molecule. Similar patterns of myosin phosphorylation are obtained in both the absence and presence of skeletal muscle actin.

A bent monomeric conformation of myosin from smooth muscle.

Smooth muscle myosin filaments formed in 0.15 M KCl are depolymerized by MgATP to a 10S component, rather than to the 6S component typical of myosin monomer in high salt concentrations. This 10S species is also monomeric as determined by sedimentation equilibrium and calculated from the diffusion and sedimentation coefficients. The conformation of 10S myosin is, however, very different from that of 6S myosin, which has a flexible but extended rod. The Stokes radius and the viscosity of 10S myosin are less than those of 6S myosin, consistent with a structure in which the rod is bent. Electron microscopy of rotary-shadowed preparations confirmed that the light meromyosin region of the rod is bent back on subfragment 2, that region of the rod adjacent to the two globular heads. MgATP and dephosphorylation of the 20,000 molecular weight light chain increase the amount of 10S myosin present in 0.15 M KCl; addition of salt converts 10S myosin back to the typical 6S conformation. We conclude that smooth muscle myosin preferentially forms a bent or folded conformation instead of the extended shape usually associated with skeletal muscle myosin, provided that the salt concentration is kept sufficiently low.

Calcium regulation of porcine aortic myosin.

Calcium regulation of actin-activated porcine aortic myosin MgATPase was studied. The MgATPase of the purified actomyosin was stimulated about 10-fold by 0.1 mM Ca2+. The 20,000 molecular weight light chain subunit (LC20) of myosin was phosphorylated by an endogenous kinase that required Ca2+. Half-maximal activation of both kinase and ATPase occurred at about 0.9 microM Ca2+. Phosphorylated and unphosphorylated myosins, free of actin, kinase, and phosphatase, were purified by gel filtration. The MgATPase of phosphorylated myosin was activated by rabbit skeletal muscle actin; unphosphorylated myosin was actin activated to a much lesser extent. Actin activation was maximal in the presence of Ca2+. Regulation of the aortic myosin MgATPase seems to involve both direct interaction of calcium with phosphorylated myosin and calcium activation of the myosin kinase. The MgATPase of trypsin-treated actomyosin did not require Ca2+ for full activity. The trypsin-treated actomyosin was devoid of LC20. When purified unphosphorylated aortic myosin was treated with trypsin, the LC20, was cleaved and the MgATPase, which was not appreciably actin activated before exposure to protease, was increased and was activated by skeletal muscle actin. After incubation of this light chain-depleted myosin with light chain from rabbit skeletal muscle myosin, the actin activation but not the increased activity, was abolished. Unphosphorylated LC20 seems to inhibit actin activation in this smooth muscle.

Amyloidosis of the AL type: Clinical, morphologic and biochemical aspects of the response to therapy with alkylating agents and prednisone

A patient with nephrotic syndrome was found to have amyloid deposition in the kidneys, skin and gastrointestinal tract. His serum and urine contained monoclonal λ light chains. In the serum, the light chains were found as tetramers and dimers, whereas the urinary form was primarily dimer. His marrow contained 16 per cent plasma cells at the time of diagnosis. He was treated with the alkylating agents melphalan and cyclophosphamide plus prednisone. He exhibited remarkable clinical improvement during the period of therapy concomitant with the eventual disappearance of monoclonal light chain production, marked reduction in proteinuria, increased normal serum protein production and documented reduction in tissue amyloid deposition. Biosynthetic studies were carried out with the patient's bone marrow cells obtained before, during and after cytotoxic therapy. Initially, λ-chain tetramers were noted in cytoplasmic extracts and secretions of the marrow cells in short-term tissue culture. Reduction and alkylation of the molecules precipitable with anti-light chain antiserums revealed light chain monomer and a smaller molecule of approximately 10 to 12,000 daltons in size. Experiments carried out after the completion of therapy revealed no detectable excess light chain synthesis. Similar studies in three other patients with AL fibril deposition indicated that all synthesized light chain dimers, but none produced larger polymers either intra- or extracellularly. A smaller molecule was identified in one of the three. These observations support the previously suggested association of the occurrence of high molecular weight light chain polymers in the serum with AL amyloid deposition. In addition, the clinical improvement noted in conjunction with cytotoxic therapy raises the possibility of the existence of subsets of patients with AL deposition who will respond to these agents.

Chick brain actin and myosin. Isolation and characterization.

Brain actin extracted from an acetone powder of chick brains was purified by a cycle of polymerization-depolymerization followed by molecular sieve chromatography. The brain actin had a subunit molecular weight of 42,000 daltons as determined by co-electrophoresis with muscle actin. It underwent salt-dependent g to f transformation to form double helical actin filaments which could be "decorated" by muscle myosin subfragment 1. A critical concentration for polymerization of 1.3 microM was determined by measuring either the change in viscosity or absorbance at 232 nm. Brain actin was also capable of stimulating the ATPase activity of muscle myosin. Brain myosin was isolated from whole chick brain by a procedure involving high salt extraction, ammonium sulfate fractionation and molecular sieve chromatography. The purified myosin was composed of a 200,000-dalton heavy chain and three lower molecular weight light chains. In 0.6 M KCl the brain myosin had ATPase activity which was inhibited by Mg++, stimulated by Ca++, and maximally activated by EDTA. When dialyzed against 0.1 M KCl, the brain myosin self-assembled into short bipolar filaments. The bipolar filaments associated with each other to form long concatamers, and this association was enhanced by high concentrations of Mg++ ion. The brain myosin did not interact with chicken skeletal muscle myosin to form hybrid filaments. Furthermore, antibody recognition studies demonstrated that myosins from chicken brain, skeletal muscle, and smooth muscle were unique.

Characterization of a second myosin from Acanthamoeba castellanii

We purified a 400,000 molecular weight myosin, myosin-II, from Acanthamoeba castellanii. The sequence of ion exchange chromatography, actomyosin precipitation, actin extraction, and gel permeation chromatography yields per 100 g of cells about 11 mg of myosin-II which is 90 to 96% pure. ATPase activity is highest in the presence of Ca2+, but the enzyme is also active in EDTA provided high concentrations of K+ are present. The molecule consists of two 175,000 molecular weight heavy chains, one or two 17,500 molecular weight light chains, and two 16,500 molecular weight light chains. Myosin-II is rich in acidic residues and contains about 32 residues of cysteine/mol. The sedimentation coefficient is 5.9 S. Intrinsic viscosity is 126 cc/g. By equilibrium ultracentrifugation, the molecular weight averages depended upon the initial loading concentration in a way that suggested a 400,000 molecular weight species is in equilibrium with a 200,000 molecular weight species. By electron microscopy the molecule was seen to have two globular heads at one end of a tail 90 nm long. In KCl solutions of less than 0.25 M, the myosin-II tails self-associate to form the backbone of very small (6.6 x 205 nm) bipolar filaments with central bare zones 97 nm long. Myosin-II binds to actin filaments, forming periodic arrowhead-shaped complexes, but its Mg2+ ATPase activity is activated only 50% or less by actin. When radioactive myosin-II is incubated up to 90 min in unlabeled Acanthamoeba homogenates, it is not degraded into smaller fragments, such as the 190,000 molecular weight myosin-I. Our observations and the detailed enzymatic data presented by Maruta and Korn ((1977) J. Biol. Chem. 252, 6501-6509) argue that the smaller Acanthamoeba myosin-I (Pollard, T. D., and Korn, E. D. (1973) J. Biol. Chem, 248, 4682-2690) does not arise by fragmentation of myosin-II in the homogenate or extract.

Myoblast myosin phosphorylation is a prerequisite for actin-activation.

CYTOPLASMIC myosins are found in most non-muscle cellular systems studied to date1. In vertebrates these myosins are composed of two heavy chains of 200,000 molecular weight (MW) and four light chains, two each of 20,000 and 15,000 MW. A number of cytoplasmic myosins, as well as skeletal, smooth, and cardiac muscle myosin, can be enzymatically phosphorylated2–6. That is, those light chains of MWs 18,000–20,000, the so-called P-light chains6, can undergo phosphorylation catalysed by a myosin light chain kinase7,8. In this reaction the terminal phosphate of ATP is transferred to a specific amino acid residue on the myosin P-light chain, up to one mole of phosphate per mole of light chain9,10. A myosin light chain phosphatase that catalyses the removal of the covalently-linked phosphate group from the P-light chain, returns the myosin to the non-phosphorylated state5,11. Phosphorylation of human platelet myosin4, guinea pig vas deferens3 and chicken gizzard12,13 smooth muscle myosin results in an increase in the actin-activated ATPase activities of these myosins. Although the phosphorylation of the P-light chain of rabbit skeletal muscle myosin has been described10, no functional change in this myosin's activity due to phosphorylation has been demonstrated11. To study the role of myosin phosphorylation during muscle cell differentiation, we investigated precursor cells to skeletal muscle, namely proliferative myoblasts. The Yaffe L-5 810 line of cloned proliferative rat myoblasts is an actively dividing cell line in culture which under the appropriate conditions can form myofibrils. We now report: (1) that myosin isolated from proliferative myoblasts contains light chains of 20,000 and 15,000 MW, (2) the 20,000 MW light chain can be phosphorylated by an endogenous myosin light chain kinase14, (3) the proliferative myoblast myosin light chain kinase does not require calcium ions for activity, and (4) phosphorylation is a pre-requisite for actin activation of the myosin's ATPase activity.

Light chains from slow-twitch muscle myosin.

Myosin light chains have been isolated from slow-twitch soleus muscles of rabbit and cat. Two chemically related light chains of molecular weight about 22 000 have been identified from their thiol sequences in each species, and these have been further characterized by amino acid analysis and peptide mapping studies. These light chains are related to the alkali light chains of rabbit fast-twitch muscles and to the larger cardiac light chain from bovine heart muscle. The presence of two chemically related but phenotypically distinct light chains within single muscles suggests the presence of myosin isoenzyme or that the myosin molecule has a different light chain associated with each subfragment-1 head. Although the stoichiometry of these two light chains had not been determined, the former of these two conclusions is favoured by analogy with experiments on fast-twitch myosins. In addition to these related light chains, soleus muscle myosin, like fast-twitch myosins, contains a third light chain of about 19 000 molecular weight. Unlike the corresponding light chain of rabbit fast-twitch myosin, this 19 000-Mr light chain contrains no cysteine residues. The distribution of thiol peptides together with the characteristic mobilities of these light chains on polyacrylamide gels in the presence of sodium dodecyl sulphate provides a 'fingerprint' of myosins from different muscle types. This has been used to look for the presence of fast-twitch myosin in soleus muscle, the results showing both rabbit and cat soleus muscles are homogeneous in their myosin type within the sensitivity of detection of the methods. These techniques have also been used to confirm the reciprocal transformation of light chains in myosins isolated from cross-reinnervated muscles. Finally the relationships between these criteria for different myosin types and histochemical procedures for muscle fibre typing are discussed.

Subunit location of sulfhydryl groups of myosin labeled with a purine disulfide analog of adenosine triphosphate.

A purine disulfide analog of ATP, 6,6'-dithiobis(inosinyl imidodiphosphate), forms mixed disulfides with cysteine residues at what are believed to be ATP regulatory sites of myosin. Blocking these sites causes inactivation of the ATPase activity at the active sites. Two cysteine residues per head are specifically modifed by this disulfide analog. The thiopurine nucleotides can be stoichiometrically displaced from myosin by [14-C]cyanide to give a more stable thiocyanato derivative of the enzyme. [14-C]Thiocyanatomyosin (3.7 14-CN/myosin) was dissociated in 4 M urea and the individual subunits were isolated. The heavy chains each had 0.78 14-CN bound per 200,000 molecular weight unit. The light chain with molecular weight of 20,700 had 1.00 14-CN bound and the 16,500 molecular weight light chain had 0.65 14-CN bound. The two 19,000 molecular weight light chains were not labeled. The two labeled light chains have only a single cysteine which is stoichiometrically modified. These two light chains show a high degree of homology and presumably perform identical functions in myosin. Their specific modification by the purine disulfide analog and their other known properties suggest that they contribute directly to the ATP regulatory sites and may, in fact, function as regulatory subunits.

Synthesis of a carboxyl-terminal (constant region) fragment of the immunoglobin light chain by a mouse myeloma cell line

The MPC11 mouse myeloma cell line synthesizes not only heavy chains and light chains but also an 11,600 molecular weight light chain fragment. The fragment comprises 1% of the newly synthesized protein, compared to 8% for the complete light chain. Similar amounts of fragment are produced by a number of heavy plus light chain producing subclones, 18 independently generated light chain producing variant clones, and five independent non-producing variant clones. For both the heavy plus light chain producing and the non-producing cell types, less than 20% of the fragment appears to be secreted, while the remainder is metabolized with a half-life of 30 minutes. Radiochemical peptide analyses and radiochemical amino -terminal sequence analyses are consistent with the fragment containing most of the peptide sequences present in the carboxyl-terminal half (constant region) of the parent kappa light chain, but none of the variable region peptides. The fragments produced by a heavy plus light chain producing clone and a non-producing variant clone were identical by radiochemical peptide analysis. The results suggest that the constant region fragment may be a primary gene product, and in addition, they raise the possibility that the fragment may be specified by a gene discrete from the gene specifying a light chain.