scholarly journals Study of Basic Local Alignment Search Tool (BLAST) and Multiple Sequence Alignment (Clustal- X) of Monoclonal mice/human antibodies

2021 ◽  
Author(s):  
IVAN VITO FERRARI ◽  
Paolo PATRIZIO
Keyword(s):  
Amino Acids ◽  
Monoclonal Antibodies ◽  
Sequence Alignment ◽  
Heavy Chain ◽  
Light Chain ◽  
Light Chains ◽  
Local Alignment ◽  
Multiple Sequence ◽  
Heavy Chains ◽  
Search Tool

In this work, we have focused on the study of the Basic Local Alignment Search Tool (BLAST) and Multiple Sequence Alignment (Clustal- X) of different monoclonal mice antibodies to understand better the multiple alignments of sequences. Our strategy was to compare the light chains of multiple monoclonal antibodies to each other, calculating their identity percentage and in which amino acid portion. (See below figure 2) Subsequently, the same survey of heavy chains was carried out with the same methodology. (See below figure 3) Finally, sequence alignment between the light chain of one antibody and the heavy chain of another antibody was studied to understand what happens if chains are exchanged between antibodies. (See below figure 4) From our results of BLAST estimation alignment, we have reported that the Light Chains (Ls) of Monoclonal Antibodies in Comparison have a sequence Homology of about 60-80% and they have a part identical in sequence zone in range 100-210 residues amino acids, except ID PDB 4ISV, which it turns out to have a 40% lower homology than the others antibodies. As far as, the heavy chains (Hs) of Monoclonal Antibodies are concerned, however they tend to have a less homology of sequences, compared to lights chains consideration, equal to 60%-70% and they have an identical part in the sequence zone between 150-210 residues amino acids; with the exception of ID PDB 3I9G-3W9D antibodies that have an equal homology at 50%. ( See supporting part) Summing up: about 70-80% identity among 2 light chains of 2 antibodies, 60-70% identity between 2 heavy chains of 2 antibodies, 30% identity between the two chains of a antibody and 30% if you compare the light chain of one antibody with the heavy chain of another antibody.

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 Clathrin assembly involves a light chain-binding region.

1987 ◽  
Vol 105 (5) ◽  
pp. 2011-2019 ◽  
Author(s):  
G S Blank ◽  
F M Brodsky
Keyword(s):  
Monoclonal Antibodies ◽  
Binding Site ◽  
Heavy Chain ◽  
Light Chain ◽  
Intermediate Filament ◽  
Binding Sites ◽  
Light Chains ◽  
Binding Region ◽  
Intermediate Filament Proteins ◽  
Interaction Site

Two regions on the clathrin heavy chain that are involved in triskelion interactions during assembly have been localized on the triskelion structure. These regions were previously identified with anti-heavy chain monoclonal antibodies X19 and X35, which disrupt clathrin assembly (Blank, G. S., and F. M. Brodsky, 1986, EMBO (Eur. Mol. Biol. Organ.) J., 5:2087-2095). Antibody-binding sites were determined based on their reactivity with truncated triskelions, and were mapped to an 8-kD region in the middle of the proximal portion of the triskelion arm (X19) and a 6-kD region at the triskelion elbow (X35). The elbow site implicated in triskelion assembly was also shown to be included within a heavy chain region involved in binding the light chains and to constitute part of the light chain-binding site. We postulate that this region of the heavy chain binds to the interaction site identified on the light chains that has homology to intermediate filament proteins (Brodsky, F. M., C. J. Galloway, G. S. Blank, A. P. Jackson, H.-F. Seow, K. Drickamer, and P. Parham, 1987, Nature (Lond.), 326:203-205). These findings suggest the existence of a heavy chain site, near the triskelion elbow, which is involved in both intramolecular and intermolecular interactions during clathrin assembly.

Novel functions of clathrin light chains: clathrin heavy chain trimerization is defective in light chain-deficient yeast

1997 ◽  
Vol 110 (7) ◽  
pp. 899-910 ◽  
Author(s):  
K.M. Huang ◽  
L. Gullberg ◽  
K.K. Nelson ◽  
C.J. Stefan ◽  
K. Blumer ◽  
...  
Keyword(s):  
Heavy Chain ◽  
Light Chain ◽  
State Level ◽  
Light Chains ◽  
Chain Gene ◽  
Light Chain Gene ◽  
Heavy Chains ◽  
Clathrin Heavy Chain ◽  
Multicopy Suppressors ◽  
Delta Cells

Clathrin is a major coat protein involved in sorting and retention of proteins at the late Golgi and in endocytosis from the cell surface. The clathrin triskelion contains three heavy chains, which provide the structural backbone of the clathrin lattice and three light chains, which are thought to regulate the formation or disassembly of clathrin coats. To better understand the function of the clathrin light chain, we characterized yeast strains carrying a disruption of the clathrin light chain gene (CLC1). Light chain-deficient cells showed phenotypes similar to those displayed by yeast that have a disruption in the clathrin heavy chain gene (CHC1). In clc1-delta cells, the steady state level of the clathrin heavy chain was reduced to 20%-25% of wild-type levels and most of the heavy chain was not trimerized. If CHC1 was overexpressed in clc1-delta cells, heavy chain trimers were detected and several clc1-delta phenotypes were partially rescued. These results indicate that the light chain is important for heavy chain trimerization and the heavy chain still has some function in the absence of the light chain. In yeast, deletion of CHC1 is lethal in strains carrying the scd1-i allele, while strains carrying the scd1-v allele can survive without the heavy chain. In previous studies we isolated several multicopy suppressors of inviability of chc1-delta scd1-i cells. Surprisingly, one of these suppressors, SCD4, is identical to CLC1. Overexpression of CLC1 in viable chc1-delta scd1-v strains rescued some but not all of the phenotypes displayed by these cells. In the absence of the heavy chain, the light chain was not found in a high molecular mass complex, but still associated with membranes. These results suggest that the light chain can function independently of the clathrin heavy chain in yeast.

Evidence for the Significant Role of Immunoglobulin Light Chains in Antigen Recognition and Selection in Chronic Lymphocytic Leukemia

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 780-780
Author(s):  
Anastasia Hadzidimitriou ◽  
Nikos Darzentas ◽  
Fiona Murray ◽  
Tanja Smilevska ◽  
Eleni Arvaniti ◽  
...  
Keyword(s):  
Amino Acid ◽  
Chronic Lymphocytic Leukemia ◽  
Heavy Chain ◽  
Light Chain ◽  
Antigen Recognition ◽  
Lymphocytic Leukemia ◽  
Light Chains ◽  
Light Chain Gene ◽  
Heavy Chains ◽  
Gene Usage

Abstract The chronic lymphocytic leukemia (CLL) immunoglobulin (IG) heavy chain repertoire is known to display biased immunoglobulin variable heavy-chain (IGHV) gene usage, remarkable complementarity determining region 3 (HCDR3) stereotypy as well as distinctive somatic hypermutation (SHM) patterns, at least for subsets of cases. Our aim in the present study was to similarly investigate the IG light chain (LC) genes in terms of mutation frequency and targeting and CDR3 stereotypy to elucidate if the LC may play a significant complementary role in antigen recognition in CLL. We thus examined SHM patterns and secondary rearrangements of the IG LC gene loci in a total of 612 IGKV-J and 279 IGLV-J rearrangements from 725 patients with CLL. Firstly, we observed a highly restricted light chain gene usage in the vast majority of CLL cases with stereotyped HCDR3s. In particular, stereotyped IGHV3-21 CLL cases were characterized by a strikingly biased expression of lambda light chains utilizing the IGLV3-21 gene (36/37 cases of subset#2), whereas all 15 subset #4 cases with stereotyped IGHV4-34 IGs carried an IGKV2-30 rearrangement. In addition, subset-biased light chain CDR3 motifs were identified in groups of sequences utilizing the same IGKV or IGLV gene. For example, all 30 IGKV1-39/1D-39 light chains of subset#1 (using stereotyped IGHV1/5/7 genes) carried notably long KCDR3s (10–11 amino acids) generated by significant N region addition and characterized by the frequent introduction of a junctional proline (26/30 cases). Important differences regarding mutational load were observed in groups of sequences utilizing the same IGKV or IGLV gene and/or belonging to subsets with stereotyped B cell receptors (BCRs). In fact, significant differences were observed with regard to mutational status among groups of sequences utilizing different alleles of certain IGK/LV genes (specifically the IGKV1-5, IGLV1-51 and IGLV3-21 genes). At cohort level, the SHM patterns were typical of a canonical SHM process. A clustering of R mutations in KCDR1 was evident for all IGKV subgroups with the notable exception of the IGKV2 subgroup, which exhibited preferential targeting to the KCDR2, especially in IGKV2-30 rearrangements of cases with stereotyped IGHV4-34/IGKV2-30 BCRs (subset#4). Recurrent amino acid changes at certain positions across the entire IGKV/IGLV sequence were observed at a high frequency (27–67% of cases) in a number of stereotyped subsets, especially those expressing the IGHV3-21/IGLV3-21 BCR (subset #2) and the IGHV4-34/IGKV2-30 BCR (subset #4). Comparison with CLL LC sequences carrying heterogeneous K/LCDR3s or non-CLL LC sequences revealed that these distinct amino acid changes are greatly under-represented in such groups and appear therefore to be “subset-biased”. Finally, a significant proportion of CLL cases (63 cases; 26 kappa- and 37 lambda-expressing) with monotypic LC expression were found to carry multiple potentially functional LC rearrangements. Of note, nineteen of these 63 cases (30%) belonged to subsets with stereotyped BCRs. This finding alludes to the possibility of secondary rearrangements most likely occurring in the context of (auto)antigen-driven receptor editing, particularly in the case of stereotyped subsets. In conclusion, SHM targeting in CLL LCs appears to be just as precise and, most likely, functionally driven as in heavy chains. Secondary LC gene rearrangements and subset-biased mutations in CLL LC genes are strong indications that LCs are crucial in shaping the specificity of leukemic BCRs, in association with defined heavy chains. Therefore, CLL is characterized not only by stereotyped HCDR3 and heavy chains but, rather, by stereotyped BCRs involving both chains, which create distinctive antigen binding grooves.

scholarly journals Goat immunoglobin G. Peptide chains and terminal residues

1969 ◽  
Vol 115 (3) ◽  
pp. 371-375 ◽  
Author(s):  
D. Givol ◽  
E. Hurwitz
Keyword(s):  
Carboxylic Acid ◽  
Heavy Chain ◽  
Light Chain ◽  
Immunoglobulin G ◽  
Light Chains ◽  
Terminal Sequence ◽  
Terminal Residue ◽  
Heavy Chains ◽  
Molecular Weights ◽  
Immunoglobin G

Goat immunoglobulin G (IgG) was isolated and characterized. The molecular weights of the IgG and its heavy chains and light chains were found to be 144000, 53600 and 23000 respectively. The light chain corresponds to human L type as was shown by the absence of C-terminal S-carboxymethylcysteine and its high content of N-terminal pyrrolid-2-one-5-carboxylic acid (PCA). The major C-terminal residue of the light chain was serine and the major N-terminal dipeptide was PCA-Ala (0·6mole/mole). The major C-terminal residue of the heavy chain was glycine and the N-terminal sequence of the heavy chain is PCA-Val-Gln. This tripeptide was obtained in a 70% yield.

scholarly journals The calpactin light chain is tightly linked to the cytoskeletal form of calpactin I: studies using monoclonal antibodies to calpactin subunits.

1987 ◽  
Vol 105 (5) ◽  
pp. 2111-2121 ◽  
Author(s):  
L Zokas ◽  
J R Glenney
Keyword(s):  
Monoclonal Antibodies ◽  
Heavy Chain ◽  
Light Chain ◽  
Human Fibroblasts ◽  
Cytoskeletal Proteins ◽  
Light Chains ◽  
Western Blots ◽  
Cultured Fibroblasts ◽  
Rous Sarcoma ◽  
Sarcoma Virus

Calpactins are a family of related Ca++-regulated cytoskeletal proteins. To analyze the expression and cytoskeletal association of calpactins we raised monoclonal antibodies with specificity for the heavy or light chains of calpactin I or to calpactin II. Comparison of the tissue distribution of calpactin I heavy and light chains by Western blots revealed that these subunits are coordinately expressed. Both soluble and cytoskeletal forms of the heavy chain of calpactin I were detected in human fibroblasts whereas only a soluble pool of calpactin II was found. These two forms of the calpactin I heavy chain differed both in their state of association with the light chain and in their rate of turnover. Both the soluble pool of the calpactin I heavy chain and calpactin II turned over three to four times faster than the cytoskeletal pool of heavy and light chains. Immunofluorescence microscopy revealed that the calpactin I light chain was present exclusively in the cytoskeleton whereas the calpactin I heavy chain distribution was more diffuse. No difference in the amount of light chain or the cytoskeletal attachment of phosphorylated calpactin I heavy chain was found in Rous sarcoma virus-transformed chick embryo fibroblasts compared with their normal counterpart. The antibody to the light chain of calpactin I was microinjected into cultured fibroblasts and kidney epithelial cells. In many cases antibody clustering was observed with the concomitant aggregation of the associated calpactin I heavy chain. The distribution of fodrin and calpactin II in injected cells remained unchanged. These results are consistent with the existence of two functionally distinct pools of calpactin I which differ in their association with the cytoskeleton.

Specificity of antigenic recognition of antibody heavy chain

1966 ◽  
Vol 166 (1003) ◽  
pp. 176-187 ◽  
Keyword(s):  
Heavy Chain ◽  
Light Chain ◽  
Group Analysis ◽  
Binding Activity ◽  
Association Constants ◽  
Light Chains ◽  
Specific Information ◽  
Parent Molecule ◽  
Heavy Chains ◽  
Antibody Heavy Chain

The specificity of antigenic recognition of the component chains of purified dinitrophenyl and trinitrophenyl antibodies was examined. Heavy chains were rendered soluble at neutral pH, either by prior reaction of the parent antibodies with D,L-alanine N -carboxy anhydride, or by mixing heavy chain with light chain of non-specific IgG. The degree of homologous light chain contamination of these heavy chain preparations was found to be less than 2 %, either by immune precipitation, or by end-group analysis. Association constants of the heavy chains of both antibodies with several closely related haptenes were measured by fluorescence quenching. Heavy chains differentiated among these haptenes in the same manner as the parent antibodies, though considerable binding affinity was lost. When specific homologous light chains were added to the heavy chain preparations, association constants were increased, but without change in relative selectivity. Binding activity of light chains alone could not be measured. The heavy chain, then, appears to bear the specificity of its parent molecule. Whether or not homologous light chain contributes additional specific information with respect to antigenic recognition or simply plays a non-specific modulating role cannot be answered from these experiments.

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 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 An Atypical Immunoglobulin

Blood ◽  
1968 ◽  
Vol 32 (2) ◽  
pp. 189-204 ◽  
Author(s):  
A. F. LEWIS ◽  
D. E. BERGSAGEL ◽  
A. BRUCE-ROBERTSON ◽  
R. K. SCHACHTER ◽  
G. E. CONNELL
Keyword(s):  
Heavy Chain ◽  
Light Chain ◽  
Gel Filtration ◽  
Sedimentation Equilibrium ◽  
Light Chains ◽  
Equilibrium Studies ◽  
Heavy Chains ◽  
Molecular Weights ◽  
Plasma Cell Neoplasm ◽  
Ig G

Abstract A protein of the Ig G family has been isolated from the serum of a patient with a tentative diagnosis of a plasma cell neoplasm. The protein has a lower sedimentation constant (5.4) and a lower molecular weight (125,000) than normal immunoglobulins of the G family. The protein has heavy-chain determinants of type G and light-chain determinants of the κ-type. Heavy and light chains have been prepared by reductive cleavage followed by gel filtration. The heavy-chain preparation is homogeneous in starch gels in acidic buffer containing urea but has a faster mobility than normal Ig G heavy chains. The light-chain preparation is resolved into two components in electrophoresis, and both have slower mobility than normal Ig G light chains. The heavy- and light-chain preparations cross react with normal Ig G heavy and light chains in immunodiffusion analysis. Sedimentation equilibrium studies suggest that both the heavy and light chains have lower molecular weights than their normal counterparts.

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