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.

scholarly journals Detection of in vitro and in vivo cleavage of high molecular weight kininogen in human plasma by immunoblotting with monoclonal antibodies

Blood ◽  
1986 ◽  
Vol 68 (2) ◽  
pp. 455-462 ◽  
Author(s):  
M Berrettini ◽  
B Lammle ◽  
T White ◽  
MJ Heeb ◽  
HP Schwarz ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Human Plasma ◽  
Heavy Chain ◽  
Light Chain ◽  
Plasma Samples ◽  
Single Chain ◽  
Sds Page ◽  
Immunoblotting Technique

Abstract Purified human high-mol-wt kininogen (HMWK), the cofactor of the contact phase of blood coagulation, migrated as a single band (approximately 110,000 mol wt) in a continuous buffer sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), but appeared as two separated bands (approximately 120,000 and 105,000 mol wt) when analyzed in a discontinuous buffer SDS-PAGE system. After elution from SDS polyacrylamide gels, each of the two bands showed coagulant activity. Six murine monoclonal antibodies (Mabs) against HMWK were produced and purified. In immunoblotting studies, three Mabs bound to the isolated alkylated heavy chain and one to the alkylated light chain of HMWK, whereas the remaining two bound only to the single-chain or unreduced two-chain molecule. None of the Mabs inhibited the clotting activity of HMWK or its binding to kaolin. Two of the Mabs, one directed against the light chain and one against the heavy chain, were used as specific probes to study HMWK in plasma samples using an immunoblotting technique. The anti-light chain Mab identified two distinct bands (approximately 120,000 and approximately 105,000 mol wt) in normal human plasma, but not in plasma from patients with hereditary HMWK deficiency. The anti-heavy chain Mab detected two additional bands (approximately 60,000 and approximately 54,000 mol wt) corresponding to low-mol-wt kininogen (LMWK) in normal plasma. A sensitive and specific quantitative immunoblotting assay of HMWK antigen in plasma was developed. Moreover, the immunoblotting technique with the anti-light chain Mab was used to detect the cleavage of HMWK in plasma samples after in vitro or in vivo activation of the contact system. The anti- light chain Mab demonstrated in vivo activation and cleavage of HMWK during an angioedema attack in a patient with hereditary angioedema and C1-inhibitor deficiency.

scholarly journals Detection of in vitro and in vivo cleavage of high molecular weight kininogen in human plasma by immunoblotting with monoclonal antibodies

Blood ◽  
1986 ◽  
Vol 68 (2) ◽  
pp. 455-462 ◽  
Author(s):  
M Berrettini ◽  
B Lammle ◽  
T White ◽  
MJ Heeb ◽  
HP Schwarz ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Human Plasma ◽  
Heavy Chain ◽  
Light Chain ◽  
Plasma Samples ◽  
Single Chain ◽  
Sds Page ◽  
Immunoblotting Technique

Purified human high-mol-wt kininogen (HMWK), the cofactor of the contact phase of blood coagulation, migrated as a single band (approximately 110,000 mol wt) in a continuous buffer sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), but appeared as two separated bands (approximately 120,000 and 105,000 mol wt) when analyzed in a discontinuous buffer SDS-PAGE system. After elution from SDS polyacrylamide gels, each of the two bands showed coagulant activity. Six murine monoclonal antibodies (Mabs) against HMWK were produced and purified. In immunoblotting studies, three Mabs bound to the isolated alkylated heavy chain and one to the alkylated light chain of HMWK, whereas the remaining two bound only to the single-chain or unreduced two-chain molecule. None of the Mabs inhibited the clotting activity of HMWK or its binding to kaolin. Two of the Mabs, one directed against the light chain and one against the heavy chain, were used as specific probes to study HMWK in plasma samples using an immunoblotting technique. The anti-light chain Mab identified two distinct bands (approximately 120,000 and approximately 105,000 mol wt) in normal human plasma, but not in plasma from patients with hereditary HMWK deficiency. The anti-heavy chain Mab detected two additional bands (approximately 60,000 and approximately 54,000 mol wt) corresponding to low-mol-wt kininogen (LMWK) in normal plasma. A sensitive and specific quantitative immunoblotting assay of HMWK antigen in plasma was developed. Moreover, the immunoblotting technique with the anti-light chain Mab was used to detect the cleavage of HMWK in plasma samples after in vitro or in vivo activation of the contact system. The anti- light chain Mab demonstrated in vivo activation and cleavage of HMWK during an angioedema attack in a patient with hereditary angioedema and C1-inhibitor deficiency.

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.

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.

scholarly journals Clathrin β-light chain of rat liver coated vesicles is phosphorylated in vitro and in vivo

FEBS Letters ◽  
1987 ◽  
Vol 220 (1) ◽  
pp. 143-148 ◽  
Author(s):  
Benoit Cantournet ◽  
Claudine Creuzet ◽  
Odile Komano ◽  
Jacques Loeb
Keyword(s):  
Rat Liver ◽  
Light Chain ◽  
Coated Vesicles

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 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.

A Molecular Approach to Immunoscintigraphy: A Study of the T-Antigen Conformation on the Surface of Tumors

1987 ◽  
Vol 26 (01) ◽  
pp. 1-6 ◽  
Author(s):  
S. Selvaraj ◽  
M. R. Suresh ◽  
G. McLean ◽  
D. Willans ◽  
C. Turner ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Tumor Cell ◽  
T Antigen ◽  
Human Carcinomas ◽  
Animal Tumor ◽  
Synthetic Antigens

The role of glycoconjugates in tumor cell differentiation has been well documented. We have examined the expression of the two anomers of the Thomsen-Friedenreich antigen on the surface of human, canine and murine tumor cell membranes both in vitro and in vivo. This has been accomplished through the synthesis of the disaccharide terminal residues in both a and ß configuration. Both entities were used to generate murine monoclonal antibodies which recognized the carbohydrate determinants. The determination of fine specificities of these antibodies was effected by means of cellular uptake, immunohistopathology and immunoscintigraphy. Examination of pathological specimens of human and canine tumor tissue indicated that the expressed antigen was in the β configuration. More than 89% of all human carcinomas tested expressed the antigen in the above anomeric form. The combination of synthetic antigens and monoclonal antibodies raised specifically against them provide us with invaluable tools for the study of tumor marker expression in humans and their respective animal tumor models.

The Development of Homologous (Canine/Anti-Canine) Antibodies in Dogs with Haemophilia A (Factor VIII Deficiency): A Ten-Year Longitudinal Study

1993 ◽  
Vol 69 (01) ◽  
pp. 021-024 ◽  
Author(s):  
Shawn Tinlin ◽  
Sandra Webster ◽  
Alan R Giles
Keyword(s):  
Factor Viii ◽  
Canine Model ◽  
Significant Inhibition ◽  
Human Condition ◽  
Haemophilia A ◽  
Variable Expression ◽  
The Family ◽  
Over Time

SummaryThe development of inhibitors to factor VIII in patients with haemophilia A remains as a serious complication of replacement therapy. An apparently analogous condition has been described in a canine model of haemophilia A (Giles et al., Blood 1984; 63:451). These animals and their relatives have now been followed for 10 years. The observation that the propensity for inhibitor development was not related to the ancestral factor VIII gene has been confirmed by the demonstration of vertical transmission through three generations of the segment of the family related to a normal (non-carrier) female that was introduced for breeding purposes. Haemophilic animals unrelated to this animal have not developed functionally significant factor VIII inhibitors despite intensive factor VIII replacement. Two animals have shown occasional laboratory evidence of factor VIII inhibition but this has not been translated into clinical significant inhibition in vivo as assessed by clinical response and F.VIII recovery and survival characteristics. Substantial heterogeneity of inhibitor expression both in vitro and in vivo has been observed between animals and in individual animals over time. Spontaneous loss of inhibitors has been observed without any therapies designed to induce tolerance, etc., being instituted. There is also phenotypic evidence of polyclonality of the immune response with variable expression over time in a given animal. These observations may have relevance to the human condition both in determining the pathogenetic factors involved in this condition and in highlighting the heterogeneity of its expression which suggests the need for caution in the interpretation of the outcome of interventions designed to modulate inhibitor activity.

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