Role of antibody heavy and light chain interface residues in affinity maturation of binding to HIV envelope glycoprotein

2019 ◽  
Vol 4 (4) ◽  
pp. 737-746 ◽  
Author(s):  
Alberto Cisneros ◽  
Rachel Stecker Nargi ◽  
Erica Hammaker Parrish ◽  
Christian Marie Haliburton ◽  
Jens Meiler ◽  
...  
Keyword(s):  
Binding Affinity ◽  
Heavy Chain ◽  
Light Chain ◽  
Envelope Glycoprotein ◽  
Affinity Maturation ◽  
Antigen Binding ◽  
Glycoprotein P ◽  
Contact Residues ◽  
Hiv Envelope

Optimization of the heavy chain/light chain interface could serve as an important tool for maximizing antibody/antigen binding affinity without altering antigen contact residues.

Highly Restricted Usage of Immunoglobulin Light Chain IGKV3-20 with Stereotyped Sequence in Primary Cold-Agglutinin Disease (CAD)

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 4137-4137 ◽  
Author(s):  
Agnieszka Malecka ◽  
Gunhild Trøen ◽  
Anne Tierens ◽  
Ingunn Østlie ◽  
Ulla Randen ◽  
...  
Keyword(s):  
Bone Marrow ◽  
B Cells ◽  
Heavy Chain ◽  
Light Chain ◽  
Cold Agglutinin ◽  
Antigen Binding ◽  
Gene Rearrangements ◽  
Cold Agglutinin Disease ◽  
Immunoglobulin Light Chain ◽  
Thermal Amplitude

Abstract Primary cold agglutinin disease (CAD) is a type of hemolytic anemia mediated by anti-I autoantibodies. Patients suffer from anemia as well as circulatory problems. However, the severity of disease differs greatly between patients. We recently demonstrated that primary CAD is caused by an underlying low grade B cell lymphoproliferative disease of the bone marrow with a typical histology that is different from lymphoplasmacytic lymphoma and, accordingly, does not display the MYD88 L265P mutation (Randen et al., Haematologica, 2013). The majority of patients display circulating monoclonal antibodies encoded by the immunoglobulin heavy chain gene IGHV4-34. The disease severity does not correlate with antibody titers, but seems to be determined by the thermal amplitude, i.e., the highest temperature at which the cold agglutinin binds to the antigen. The framework region 1 of IGHV4-34 encodes for a sequence that binds to I antigen. However, this does not explain the molecular basis of disease heterogeneity. We studied 27 patients with well-characterized primary CAD and sequenced immunoglobulin heavy as well as immunoglobulin light chains to find additional consensus regions that may determine anti-I reactivity. Bone marrow aspirates, or frozen bone marrow trephine biopsies and blood from 27 patients with well-documented primary CAD were collected. Monoclonal B cells were isolated by flow sorting (FACS Aria Ilu High speed sorter, Becton Dickinson). Viable cells were detected using the forward scatter versus side scatter dot plot. Subsequently, CD45 bright events with low side scatter features representing lymphocytes, were selected. Then, CD5 positive and CD19 negative events, i.e. T cells, were gated out using a CD5 versus CD19 dot plot leaving only B cells. Finally, monoclonal B cells were selected using the immunoglobulin light chain gate, either k or l. Clonally rearranged IGH genes were detected using the Somatic Hypermutation Assay v2.0 (Invivoscribe) and were then sequenced. Immunoglobulin light chain genes (IGL) were amplified by an in-house diagnostic protocol based on Biomed-2 primers (van Dongen et al., Leukemia, 2003). All sequences were analyzed using the IMGT database (www.imgt.org). Productive IGHV4-34 gene rearrangements were identified in 22/27 patients. In 4 patients, no productive rearrangement was identified, while in one patient a productive IGHV3-23 was seen. No significant homology of complementarity determining region 3 (CDR3) regions was found between IGHV sequences. The N-glycosylation sequence within the CDR2 region, affecting antigen-binding, was mutated in 8 patients whereas no mutations were present in 7 patients and mutations in flanking residues were seen in 6 patients. The latter mutations may modulate glycosylation efficacy. Clonal rearrangement of the IGKV3-20 was detected in 16/27 patients, clonal IGKV3-15 gene rearrangements were identified in 4/27 patients whereas other IGL genes were rearranged in 4/27 patients. No clonal IGL gene rearrangement was found in 3/27 patients. Of interest, 7 of the patients with IGKV3-20 rearrangement displayed highly homologous CDR3 regions. The latter was highly associated with an un-mutated N-glycosylation sequence of the respective IGHV4-34 sequence. In conclusion, our data show that in addition to IGHV, also IGLV usage is highly restricted in CAD. Furthermore, stereotyped IGLV sequences are seen that are mutually exclusive with mutated N-glycosylation sequences in the IGHV CDR2 sequence. These data indicate that multiple regions within the immunoglobulin heavy chain as well as immunoglobulin light chain contribute to I-antigen binding. The data suggest that subtle differences in these multiple binding sequences may contribute to the differences in thermal amplitude of I antigen binding of the antibody. The highly restricted usage of IGKV3-20 provides a rationale for vaccination with IGKV3-20 proteins, known to be immunogenic and being considered for treatment in other lymphoproliferative diseases (Martorelli et al., Clin Cancer Res, 2012). Disclosures No relevant conflicts of interest to declare.

scholarly journals DEMONSTRATION OF HEAVY AND LIGHT CHAIN ANTIGENIC DETERMINANTS ON THE CELL-BOUND RECEPTOR FOR ANTIGEN

1970 ◽  
Vol 132 (6) ◽  
pp. 1233-1249 ◽  
Author(s):  
Curla S. Walters ◽  
Hans Wigzell
Keyword(s):  
Heavy Chain ◽  
Light Chain ◽  
Immune Cells ◽  
Surface Structures ◽  
High Rate ◽  
Antigenic Determinants ◽  
Antigen Binding ◽  
Memory Cells ◽  
Humoral Antibody ◽  
Heavy Chains

High-rate antibody-forming cells and immunological memory cells can be selectively retained if filtered through a column coated with relevant antigen. This trapping can be blocked if the cells are incubated with an anti-immunoglobulin serum prior to column passage. A similar blocking is not observed when cells are treated with an anti-lymphocyte serum, thereby excluding the possibility that any antibodies combining with surface structures could cause this effect. By the use of antisera specific for heavy or light chain antigens, it was possible to locate such antigens in the antigen-binding receptor areas of immune cells. Criss-cross studies using antisera specific for gamma 1 or gamma 2a heavy chains showed that the membrane receptor has the same heavy chain as will be present in the eventual product of that cell, the humoral antibody.

scholarly journals Affinity maturation is required for pathogenic monovalent IgG4 autoantibody development in myasthenia gravis

2020 ◽  
Vol 217 (12) ◽  
Author(s):  
Miriam L. Fichtner ◽  
Casey Vieni ◽  
Rachel L. Redler ◽  
Ljuvica Kolich ◽  
Ruoyi Jiang ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
Myasthenia Gravis ◽  
Crystal Structures ◽  
Binding Affinity ◽  
Somatic Mutations ◽  
Affinity Maturation ◽  
Antigen Binding ◽  
High Affinity ◽  
Specific Igg4 ◽  
Autoimmune Myasthenia

Pathogenic muscle-specific tyrosine kinase (MuSK)–specific IgG4 autoantibodies in autoimmune myasthenia gravis (MG) are functionally monovalent as a result of Fab-arm exchange. The development of these unique autoantibodies is not well understood. We examined MG patient–derived monoclonal autoantibodies (mAbs), their corresponding germline-encoded unmutated common ancestors (UCAs), and monovalent antigen-binding fragments (Fabs) to investigate how affinity maturation contributes to binding and immunopathology. Mature mAbs, UCA mAbs, and mature monovalent Fabs bound to MuSK and demonstrated pathogenic capacity. However, monovalent UCA Fabs bound to MuSK but did not have measurable pathogenic capacity. Affinity of the UCA Fabs for MuSK was 100-fold lower than the subnanomolar affinity of the mature Fabs. Crystal structures of two Fabs revealed how mutations acquired during affinity maturation may contribute to increased MuSK-binding affinity. These findings indicate that the autoantigen drives autoimmunity in MuSK MG through the accumulation of somatic mutations such that monovalent IgG4 Fab-arm–exchanged autoantibodies reach a high-affinity threshold required for pathogenic capacity.

scholarly journals Novel Features of the Light Chain of Microtubule-associated Protein MAP1B: Microtubule Stabilization, Self Interaction, Actin Filament Binding, and Regulation by the Heavy Chain

1998 ◽  
Vol 143 (3) ◽  
pp. 695-707 ◽  
Author(s):  
Martin Tögel ◽  
Gerhard Wiche ◽  
Friedrich Propst
Keyword(s):  
Actin Filament ◽  
Heavy Chain ◽  
Light Chain ◽  
Regulatory Subunit ◽  
Light Chains ◽  
Microtubule Stabilization ◽  
Microtubule Stability ◽  
Control Light ◽  
Chain Interaction

Previous studies on the role of microtubule-associated protein 1B (MAP1B) in adapting microtubules for nerve cell-specific functions have examined the activity of the entire MAP1B protein complex consisting of heavy and light chains and revealed moderate effects on microtubule stability. Here we have analyzed the effects of the MAP1B light chain in the absence or presence of the heavy chain by immunofluorescence microscopy of transiently transfected cells. Distinct from all other MAPs, the MAP1B light chain–induced formation of stable but apparently flexible microtubules resistant to the effects of nocodazole and taxol. Light chain activity was inhibited by the heavy chain. In addition, the light chain was found to harbor an actin filament binding domain in its COOH terminus. By coimmunoprecipitation experiments using epitope-tagged fragments of MAP1B we showed that light chains can dimerize or oligomerize. Furthermore, we localized the domains for heavy chain–light chain interaction to regions containing sequences homologous to MAP1A. Our findings assign several crucial activities to the MAP1B light chain and suggest a new model for the mechanism of action of MAP1B in which the heavy chain might act as the regulatory subunit of the MAP1B complex to control light chain activity.

scholarly journals Clathrin light chain directs endocytosis by influencing the binding of the yeast Hip1R homologue, Sla2, to F-actin

2011 ◽  
Vol 22 (19) ◽  
pp. 3699-3714 ◽  
Author(s):  
Douglas R. Boettner ◽  
Helena Friesen ◽  
Brenda Andrews ◽  
Sandra K. Lemmon
Keyword(s):  
Heavy Chain ◽  
Light Chain ◽  
Deletion Mutation ◽  
Actin Binding ◽  
Synthetic Genetic Array ◽  
Array Analysis ◽  
Clathrin Heavy Chain ◽  
N Terminus ◽  
Mechanistic Basis

The role of clathrin light chain (CLC) in clathrin-mediated endocytosis is not completely understood. Previous studies showed that the CLC N-terminus (CLC-NT) binds the Hip1/Hip1R/Sla2 family of membrane/actin–binding factors and that overexpression of the CLC-NT in yeast suppresses endocytic defects of clathrin heavy-chain mutants. To elucidate the mechanistic basis for this suppression, we performed synthetic genetic array analysis with a clathrin CLC-NT deletion mutation (clc1-Δ19-76). clc1-Δ19-76 suppressed the internalization defects of null mutations in three late endocytic factors: amphiphysins (rvs161 and rvs167) and verprolin (vrp1). In actin sedimentation assays, CLC binding to Sla2 inhibited Sla2 interaction with F-actin. Furthermore, clc1-Δ19-76 suppression of the rvs and vrp phenotypes required the Sla2 actin-binding talin-Hip1/R/Sla2 actin-tethering C-terminal homology domain, suggesting that clc1-Δ19-76 promotes internalization by prolonging actin engagement by Sla2. We propose that CLC directs endocytic progression by pruning the Sla2-actin attachments in the clathrin lattice, providing direction for membrane internalization.

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