scholarly journals BiP and Immunoglobulin Light Chain Cooperate to Control the Folding of Heavy Chain and Ensure the Fidelity of Immunoglobulin Assembly

1999 ◽  
Vol 10 (7) ◽  
pp. 2209-2219 ◽  
Author(s):  
Young-Kwang Lee ◽  
Joseph W. Brewer ◽  
Rachel Hellman ◽  
Linda M. Hendershot
Keyword(s):  
Heavy Chain ◽  
Light Chain ◽  
Protein Complexes ◽  
Critical Role ◽  
Light Chains ◽  
Heavy Chains ◽  
Chain Complexes ◽  
Chain Synthesis

The immunoglobulin (Ig) molecule is composed of two identical heavy chains and two identical light chains (H2L2). Transport of this heteromeric complex is dependent on the correct assembly of the component parts, which is controlled, in part, by the association of incompletely assembled Ig heavy chains with the endoplasmic reticulum (ER) chaperone, BiP. Although other heavy chain-constant domains interact transiently with BiP, in the absence of light chain synthesis, BiP binds stably to the first constant domain (CH1) of the heavy chain, causing it to be retained in the ER. Using a simplified two-domain Ig heavy chain (VH-CH1), we have determined why BiP remains bound to free heavy chains and how light chains facilitate their transport. We found that in the absence of light chain expression, the CH1 domain neither folds nor forms its intradomain disulfide bond and therefore remains a substrate for BiP. In vivo, light chains are required to facilitate both the folding of the CH1 domain and the release of BiP. In contrast, the addition of ATP to isolated BiP–heavy chain complexes in vitro causes the release of BiP and allows the CH1 domain to fold in the absence of light chains. Therefore, light chains are not intrinsically essential for CH1 domain folding, but play a critical role in removing BiP from the CH1 domain, thereby allowing it to fold and Ig assembly to proceed. These data suggest that the assembly of multimeric protein complexes in the ER is not strictly dependent on the proper folding of individual subunits; rather, assembly can drive the complete folding of protein subunits.

scholarly journals Immunoglobulin heavy chain and binding protein complexes are dissociated in vivo by light chain addition.

1990 ◽  
Vol 111 (3) ◽  
pp. 829-837 ◽  
Author(s):  
L M Hendershot
Keyword(s):  
Heavy Chain ◽  
Light Chain ◽  
Secretory Pathway ◽  
Protein Transport ◽  
Protein Complexes ◽  
Binding Protein ◽  
Immunoglobulin Heavy Chain ◽  
Light Chains ◽  
Heavy Chains

Immunoglobulin heavy chain binding protein (BiP, GRP78) associates stably with the free, nonsecreted Ig heavy chains synthesized by Abelson virus transformed pre-B cell lines. In cells synthesizing both Ig heavy and light chains, the Ig subunits assemble rapidly and are secreted. Only incompletely assembled Ig molecules can be found bound to BiP in these cells. In addition to Ig heavy chains, a number of mutant and incompletely glycosylated transport-defective proteins are stably complexed with BiP. When normal proteins are examined for combination with BiP, only a small fraction of the intracellular pool of nascent, unfolded, or unassembled proteins can be found associated. It has been difficult to determine whether these BiP-associated molecules represent assembly intermediates which will be displaced from BiP and transported from the cell, or whether these are aberrant proteins that are ultimately degraded. In order for BiP to monitor and aid in normal protein transport, its association with these proteins must be reversible and the released proteins should be transport competent. In the studies described here, transient heterokaryons were formed between a myeloma line producing BiP-associated heavy chains and a myeloma line synthesizing the complementary light chain. Introduction of light chain synthesis resulted in assembly of prelabeled heavy chains with light chains, displacement of BiP from heavy chains, and secretion of Ig into the culture supernatant. These data demonstrate that BiP association can be reversible, with concordant release of transportable proteins. Thus, BiP can be considered a component of the exocytic secretory pathway, regulating the transport of both normal and abnormal proteins.

An Essential Role of the Factor VIII Light Chain in Facilitating Heavy Chain Secretion.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 4034-4034
Author(s):  
Lingxia Chen ◽  
Juan Li ◽  
Hui Lu ◽  
Haiyan Jiang ◽  
Rita Sarkar ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Factor Viii ◽  
Heavy Chain ◽  
Light Chain ◽  
Hek293 Cells ◽  
Light Chains ◽  
Trans Splicing ◽  
In Cis

Abstract Blood coagulation Factor VIII (FVIII) is secreted as a heterodimer consisting of a heavy and light chain. Both in vitro and in vivo studies have demonstrated that these chains can be expressed independently. The expressed heavy and light chains can reassociate with recovery of biological activities. These observations have been particularly useful in a gene therapy setting since vector packaging capacity for adeno-associated virus (AAV) is a limiting factor. However, it has been demonstrated that the FVIII heavy chain is expressed ~10–100-fold less efficiently compared to the light chain when expressed independently. Previously the FVIII F309S mutation in the context of B-domainless FVIII (FVIII-BDD) and enhanced glycosylations within the B-domain have been shown to improve factor VIII expression and secretion. However, our in vitro studies indicate that these improvements in secretion were not retained when expressing the heavy chain alone with the same modifications. Other sequences, possibly in the light chain, may facilitate secretion. To investigate this further, we designed an intein trans-splicing strategy to control the addition of light chain to the heavy chain before secretion. Using HEK293 cells, we cotransfected seperate intein light chain and intein heavy chain plasmids and compared results to single plasmid transfected cells. 48 hours post-transfection, FVIII-specific ELISA results demonstrated that cotransfection of intein heavy chain and intein light chain had a significant influence on total heavy chain secretion compared to intein heavy chain expression alone. The co-transfected intein heavy chain and intein light chain were efficiently ligated together yielding a biologically active single chain FVIII derivative as demonstrated by clotting assays and Western blot analysis. Therefore, heavy chain secretion was directly enhanced by the attachment of the light chain to the C-terminus of the heavy chain. A similar phenomenon was not found when heavy and light chains were simply co-expressed in the same cell. It suggested that light chain functioned in cis. Hydrodynamic injection of plasmids with intein heavy chain and intein light chain into hemophilia A mice led to a much higher level of FVIII secretion. The amount of functional FVIII expression reached 3–6 units/ml at peak level. In the absence of intein light chain, FVIII heavy chain secretion was approximately 100 fold less efficient in vivo. To map the key elements of FVIII light in helping FVIII secretion, we made deletion variants in the light chain. These mutants had a dominant negative effect in reducing FVIII and FVIII heavy chain secretion while increasing the level of intracellular FVIII accumulation. Collectively our results are consistent with the conclusion that the FVIII light chain plays a critical role in facilitating heavy chain secretion in cis; probably through helping FVIII heavy chain maintain correct configuration and folding. The strategy to manipulate FVIII light chain addition through intein mediated trans-splicing reaction may also be explored for human gene therapy.

An Atypical Human Immunoglobulin G with Deletions in both Heavy and Light Chains. Studies of the Conformation and the In Vitro Recombination of the Isolated Subunits

1974 ◽  
Vol 52 (7) ◽  
pp. 610-619 ◽  
Author(s):  
M. E. Percy ◽  
K. J. Dorrington
Keyword(s):  
Heavy Chain ◽  
Light Chain ◽  
Hypervariable Region ◽  
Light Chains ◽  
Constant Region ◽  
Myeloma Protein ◽  
Variable Regions ◽  
Heavy Chains ◽  
Amino Terminal

Both the light and gamma (heavy) chains of IgG(Sac) contain extensive deletions in their variable regions. The deletion in the light chain is internal (residues 18–88), whereas the deletion in the heavy chain is amino-terminal (residues 1–102). The hypervariable region just preceding the beginning of the constant region in other heavy chains (residues 103–115) is amino-terminal in heavy chain(Sac). In 4 mM acetate, pH 5.4, heavy chain(Sac) is dimeric like normal gamma chains, whereas light chain(Sac) is monomeric. Isolated light and heavy chains of IgG(Sac) recombine in vitro with each other and also with the heavy and light chains from a typical human IgG1-K myeloma protein, but not in a fashion entirely typical of other human gamma and light chains. These studies support the concept that non-covalent forces between the variable regions of the light and heavy chains are important in the assembly of the immunoglobulin molecule; and in view of the weak interaction between the constant region of light chain and heavy chain observed previously, our data suggest that there are points of contact between the hypervariable region of the gamma chain (residues 103–115) and the variable region of the light chain.

scholarly journals Immunoglobulin M biosynthesis. Production of intermediates and excess of light chain in mouse myeloma MOPC 104E

1971 ◽  
Vol 123 (4) ◽  
pp. 635-641 ◽  
Author(s):  
R. M. E. Parkhouse
Keyword(s):  
Heavy Chain ◽  
Light Chain ◽  
Gradient Centrifugation ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sucrose Density Gradient ◽  
Immunoglobulin M ◽  
Sucrose Density Gradient Centrifugation ◽  
Chain Synthesis

Immunoglobulin M (IgM) biosynthesis was studied with mouse plasma-cell tumour MOPC 104E as a model system. Cell suspensions prepared from solid tumours were incubated in vitro with [3H]leucine; the radioactivity incorporated into intracellular and secreted proteins was analysed by sucrose-density-gradient centrifugation and polyacrylamide-gel electrophoresis. The tumour secretes IgM and light chains. ‘Pulse–chase’ experiments indicated average secretion times of 1.5h for light chain and 2.5h for IgM. The order of disulphide-bond assembly within the cell was shown to be heavy chain+light chain → heavy chain–light chain intermediate → IgMs. The 7S subunit (IgMs) was polymerized into IgM just before or at the time of secretion. Measurements of heavy-chain/light-chain radioactivity ratios in intracellular HL and IgMs and secreted IgM demonstrated the existence of a light-chain pool participating in IgM biosynthesis. The size of the light-chain pool, together with analysis of clones isolated in vivo, suggested that the tumour contains cells in which light-chain synthesis is in excess of heavy-chain production.

In vitro and in vivo expression of a nephritogenic Ig heavy chain determinant: Pathogenic autoreactivity requires permissive light chains

2001 ◽  
Vol 79 (3) ◽  
pp. 222-230 ◽  
Author(s):  
Brenda G Cooperstone ◽  
Mohammed M Rahman ◽  
Earl H Rudolph ◽  
Mary H Foster
Keyword(s):  
Heavy Chain ◽  
Light Chains ◽  
In Vivo Expression ◽  
Ig Heavy Chain

Control of AMP deaminase 1 binding to myosin heavy chain

1998 ◽  
Vol 275 (3) ◽  
pp. C870-C881 ◽  
Author(s):  
Ichiro Hisatome ◽  
Takayuki Morisaki ◽  
Hiroshi Kamma ◽  
Takako Sugama ◽  
Hiroko Morisaki ◽  
...  
Keyword(s):  
Myosin Heavy Chain ◽  
Heavy Chain ◽  
Sequence Similarity ◽  
Critical Role ◽  
Energy Charge ◽  
Adenylate Energy Charge ◽  
Amp Deaminase ◽  
Amino Terminal

AMP deaminase (AMPD) plays a central role in preserving the adenylate energy charge in myocytes following exercise and in producing intermediates for the citric acid cycle in muscle. Prior studies have demonstrated that AMPD1 binds to myosin heavy chain (MHC) in vitro; binding to the myofibril varies with the state of muscle contraction in vivo, and binding of AMPD1 to MHC is required for activation of this enzyme in myocytes. The present study has identified three domains in AMPD1 that influence binding of this enzyme to MHC using a cotransfection model that permits assessment of mutations introduced into the AMPD1 peptide. One domain that encompasses residues 178–333 of this 727-amino acid peptide is essential for binding of AMPD1 to MHC. This region of AMPD1 shares sequence similarity with several regions of titin, another MHC binding protein. Two additional domains regulate binding of this peptide to MHC in response to intracellular and extracellular signals. A nucleotide binding site, which is located at residues 660–674, controls binding of AMPD1 to MHC in response to changes in intracellular ATP concentration. Deletion analyses demonstrate that the amino-terminal 65 residues of AMPD1 play a critical role in modulating the sensitivity to ATP-induced inhibition of MHC binding. Alternative splicing of the AMPD1 gene product, which alters the sequence of residues 8–12, produces two AMPD1 isoforms that exhibit different MHC binding properties in the presence of ATP. These findings are discussed in the context of the various roles proposed for AMPD in energy production in the myocyte.

scholarly journals Clathrin structure characterized with monoclonal antibodies. II. Identification of in vivo forms of clathrin.

1985 ◽  
Vol 101 (6) ◽  
pp. 2055-2062 ◽  
Author(s):  
F M Brodsky
Keyword(s):  
Monoclonal Antibodies ◽  
Heavy Chain ◽  
Light Chain ◽  
Coated Vesicles ◽  
Cell Lysates ◽  
Over Time

Clathrin was isolated from detergent-solubilized, biosynthetically radiolabeled cells by immunoprecipitation with anti-clathrin monoclonal antibodies. Immunoprecipitates obtained after pulse-chase labeling demonstrated that after biosynthesis the LCa light chain of clathrin could be found either complexed to heavy chain or in a free pool (not associated with heavy chain) which decreased steadily over time. More than half of the free LCa disappeared within the first hour after biosynthesis, but some was still detectable after several hours. Incorporation of clathrin LCa light chain and heavy chain into coated vesicles was coordinate and increased up to 4 h after biosynthesis. Comparison of these kinetics suggested that once incorporated into coated vesicles, LCa and heavy chain did not dissociate, even during depolymerization of the vesicle. There was also little apparent degradation of clathrin found in coated vesicles for up to 22 h after biosynthesis. Immunoprecipitation with anti-clathrin monoclonal antibodies was carried out after fractionation of continuously radiolabeled cell lysates using two different sizing columns. These experiments indicated that the triskelion form of clathrin that has been isolated from coated vesicles in vitro also exists in vivo. They also confirmed the existence of a transient but detectable pool of newly synthesized free LCa light chain.

Inhibitory ADAMTS13 Monoclonal Autoantibodies Cloned from TTP Patients Show Restricted Gene Usage, Inhibit Murine ADAMTS13, and Are Blocked by Rabbit Anti-Idiotypic Antibodies.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 92-92 ◽  
Author(s):  
Don Siegel ◽  
Eric Ostertag
Keyword(s):  
Immune Response ◽  
Phage Display ◽  
Heavy Chain ◽  
Light Chain ◽  
Light Chains ◽  
Chain Gene ◽  
Human Mabs ◽  
Heavy Chains ◽  
Phage Display Libraries ◽  
Pathogenic Antibodies

Abstract Thrombotic thrombocytopenic purpura (TTP) is a potentially fatal disorder often associated with autoantibody inhibition of ADAMTS13, a VWF-cleaving protease. Autoantibodies decrease ADAMTS13 activity resulting in accumulation of “unusually” large VWF multimers that mediate platelet thrombosis. To better understand the role autoantibodies play in disease pathogenesis, as well as to develop more specific methods for diagnosis and therapy, it is necessary to characterize pathogenic antibodies on a molecular level, something not possible through analysis of polyclonal patient antisera. The ability to clone large repertoires of patient monoclonal autoantibodies (mAbs) using phage display offers a unique opportunity to address this issue. Three patient (Pt) antibody phage display libraries were created from either splenocytes (Pt1) or peripheral blood lymphocytes (Pt2, Pt3) of individuals with acquired TTP. ADAMTS13-specific mAbs were isolated by panning against recombinant ADAMTS13. Unique clones were identified by DNA sequencing, and their ability to interact with ADAMTS13 was characterized. After antigen selection of Pt1 library, 56 mAbs were randomly-selected from panning rounds 2 through 4 and 68% were found to comprise heavy chains encoded by VH1-69 paired with a VL3 family lambda light chain (3h or 3m). The remaining mAbs comprised heavy chains from the VH1, 3, or 4 families usually paired with kappa light chains. For Pt2 and Pt3 libraries, there was an identical pattern of genetic restriction in immune response to ADAMTS13, i.e. 16 of 24 mAbs (Pt2) and 27 of 27 mAbs (Pt3) were encoded by VH1-69 heavy chains and VL3 family lambda light chains. Though nearly all mAbs were unique, common CDR3 regions among some of the mAbs provided evidence of B-cell clonal expansion and somatic mutation. Though all mAbs bound to ADAMTS13 irrespective of genetic origin, mAbs comprising a VH1-69 heavy chain paired with a VL3 light chain inhibited ADAMTS13 using the FRET-VW73 assay while mAbs comprising a VH1-69 paired with a kappa light chain or comprising non-VH1-69 heavy chains did not inhibit ADAMTS13, with only two exceptions. MAb binding to ADAMTS13 was blocked by preincubation with normal human or murine plasma, but much less so by plasma from TTP patients or ADAMTS13 knockout mice suggesting crossreactivity with mouse ADAMTS13. Certain human mAbs inhibited cleavage of FRET-VWF73 by mouse ADAMTS13 and also inhibited ADAMTS13 in vivo after injection into the internal jugular vein of mice. Rabbit anti-idiotypic antibodies raised against mAb 416, a prototypical VH1-69-encoded mAb, blocked 416’s ability to inhibit human ADAMTS13. Taken together, the cloning and analyses of a large cohort of ADAMTS13 inhibitory autoantibodies derived from 3 unrelated individuals with acquired TTP revealed a genetically restricted immune response. This feature, if common among TTP patients, offers a potential therapeutic target for treatment of TTP, e.g. selective deletion of B-cells utilizing the VH1-69 heavy chain gene. Furthermore, crossreactivity of some human mAbs with murine ADAMTS13 provides a mouse model of acquired ADAMTS13 deficiency that may prove useful for determining the role of autoantibodies in the pathogenesis of TTP, particularly in the context of additional factors (e.g. environmental) that may be required to trigger the disease. Finally, anti-idiotypic mAbs, currently being cloned from rabbit phage display libraries, may help identify pathogenic antibodies in patient plasma and/or lead to novel therapeutic approaches.

scholarly journals Identification of a Ca(2+)-binding light chain within Chlamydomonas outer arm dynein

1995 ◽  
Vol 108 (12) ◽  
pp. 3757-3764 ◽  
Author(s):  
S.M. King ◽  
R.S. Patel-King
Keyword(s):  
Heavy Chain ◽  
Light Chain ◽  
Dynein Light Chain ◽  
Binding Studies ◽  
Significant Similarity ◽  
Helix Loop Helix ◽  
Ef Hand ◽  
Chain Sequence

We describe here the molecular cloning of the M(r) 18,000 dynein light chain from the outer arm of Chlamydomonas flagella. In vivo, this molecule is directly associated with the gamma dynein heavy chain. Sequence analysis indicates that this light chain is a novel member of the calmodulin superfamily of Ca2+ binding regulatory proteins; this molecule is 42, 37 and 36% identical to calmodulin, centrin/caltractin and troponin C, respectively, and also shows significant similarity to myosin light chains. Although four helix-loop-helix elements are evident, only two conform precisely to the EF hand consensus and are therefore predicted to bind Ca2+ in vivo. In vitro Ca2+ binding studies indicate that this dynein light chain (expressed as a C-terminal fusion with maltose binding protein) has at least one functional Ca2+ binding site with an apparent affinity for Ca2+ of approximately 3 × 10(−5) M. Within the Chlamydomonas flagellum, the transition from an assymmetric to a symmetric waveform (which implies an alteration in dynein activity) is mediated by an increase in intraflagellar Ca2+ from 10(−6) to 10(−1) M; this transition is altered in mutants that lack the outer arm. The data presented here suggest that a Ca(2+)-dependent alteration in the interaction of this dynein light chain with the motor containing heavy chain may affect outer arm function during flagellar reversal.

Recombinant Rat Protein C: Comparative Studies of Structure, Function and Synthesis with Plasma Protein C

1994 ◽  
Vol 71 (01) ◽  
pp. 054-061 ◽  
Author(s):  
Mayumi Ono ◽  
Hiroyuki Fujiwara ◽  
Takaaki Okafuji ◽  
Tomoko Enjoh ◽  
Katsuhiko Nawa
Keyword(s):  
Heavy Chain ◽  
Light Chain ◽  
Protein C ◽  
Rat Hepatocytes ◽  
Rat Plasma ◽  
Chain Molecule ◽  
Dependent Manner ◽  
Single Chain

SummaryIn order to elucidate the role of protein C (PC) in the rat, we expressed, purified, and characterized recombinant rat PC. The purified recombinant rat PC was 70–90% two-chain (41 kDa heavy chain; 22 and 23 kDa light chain) and 10–30% single-chain (61 kDa). Amino acid analysis confirmed the presence of 10 moles of γ-carboxyglutamic acid residues per mol of protein. For comparison, plasma rat PC was purified from a barium citrate precipitate using similar method. Plasma rat PC was a two-chain form (41 kDa heavy chain; 22 kDa light chain) with no detectable single-chain nor 23 kDa light chain. For determination of the in vitro secreted species, primary cultured rat hepatocytes were incubated for 6 h with methionine-free MEM containing vitamin K1, aprotinin, and [35S]methionine. The supernatant was immunoprecipitated and analyzed by SDS-PAGE followed by autoradiography. Approximately 90% of the PC radioactivity migrated as a two-chain molecule. These results indicate that rat PC is secreted mainly as a two-chain molecule from the liver. PROTAC-activated forms of recombinant rat PC, plasma rat PC, and plasma human PC hydrolyzed the S-2366 chromogenic substrate at the same rate Recombinant rat PC was also activated by the thrombin-thrombomodulin complex at a rate similar to plasma lat PC. The anticoagulant activities of the three activated PCs were examined in rat plasma. Both recombinant and plasma rat PC prolonged the activated partial thromboplastin time in a dose-dependent manner, but plasma human PC was less effective. These results suggest that recombinant rat PC is applicable for in vivo thrombosis studies in the rat.

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