Level of galactosylation determines cryoglobulin activity of murine IgG3 monoclonal rheumatoid factor

Blood ◽  
2002 ◽  
Vol 99 (8) ◽  
pp. 2922-2928 ◽  
Author(s):  
Aki Kuroki ◽  
Yasuhiro Kuroda ◽  
Shuichi Kikuchi ◽  
Frédéric Lajaunias ◽  
Thierry Fulpius ◽  
...  
Keyword(s):  
Rheumatoid Factor ◽  
Heavy Chain ◽  
Glycosylation Site ◽  
Structural Basis ◽  
Side Chains ◽  
Murine Lupus ◽  
Marked Contrast ◽  
Autoimmune Syndrome ◽  
Molecular Variants ◽  
Charged Residues

Abstract Autoantibodies of the cryoprecipitating IgG3 isotype have been shown to play a significant role in the development of murine lupus–like autoimmune syndrome. At present, the structural basis of IgG3 cryoprecipitation and its role in autoantibody pathogenicity remain to be defined. Using molecular variants of an IgG3 monoclonal rheumatoid factor, 6-19, derived from an autoimmune MRL-Faslpr mouse, we have investigated the implication of charged residues in the heavy-chain variable (VH) region, potential CH3-linked oligosaccharides, and galactosylation of CH2-linked oligosaccharides in its cryoglobulin activity. The cryoglobulin activity of the IgG3 6-19 mutant bearing more negatively charged residues at VH 6 and 23 was found to be reduced but still highly significant, whereas that of the mutant lacking a potential CH3 glycosylation site remained unchanged. In marked contrast, IgG3 6-19 variants obtained from 6-19 heavy-chain transgenic mice displayed barely detectable cryoglobulin activity associated with an increased level of galactosylation in the CH2 oligosaccharide side chains. Thus, our data strongly suggest that the cryoglobulin activity of IgG3 6-19 autoantibody is critically determined by levels of galactosylation in the CH2 oligosaccharide side chains, whereas VH residues play a secondary role in 6-19 IgG3 cryoglobulin activity.

scholarly journals Structural basis of polyethylene glycol recognition by antibody

2020 ◽  
Vol 27 (1) ◽  
Author(s):  
Cheng-Chung Lee ◽  
Yu-Cheng Su ◽  
Tzu-Ping Ko ◽  
Li-Ling Lin ◽  
Chih-Ya Yang ◽  
...  
Keyword(s):  
Polyethylene Glycol ◽  
Crown Ether ◽  
Heavy Chain ◽  
Light Chain ◽  
Underlying Mechanism ◽  
Binding Mode ◽  
Structural Basis ◽  
Side Chains ◽  
Arginine Residues ◽  
Aromatic Side Chains

Abstract Background Polyethylene glycol (PEG) is widely used in industry and medicine. Anti-PEG antibodies have been developed for characterizing PEGylated drugs and other applications. However, the underlying mechanism for specific PEG binding has not been elucidated. Methods The Fab of two cognate anti-PEG antibodies 3.3 and 2B5 were each crystallized in complex with PEG, and their structures were determined by X-ray diffraction. The PEG-Fab interactions in these two crystals were analyzed and compared with those in a PEG-containing crystal of an unrelated anti-hemagglutinin 32D6-Fab. The PEG-binding stoichiometry was examined by using analytical ultracentrifuge (AUC). Results A common PEG-binding mode to 3.3 and 2B5 is seen with an S-shaped core PEG fragment bound to two dyad-related Fab molecules. A nearby satellite binding site may accommodate parts of a longer PEG molecule. The core PEG fragment mainly interacts with the heavy-chain residues D31, W33, L102, Y103 and Y104, making extensive contacts with the aromatic side chains. At the center of each half-circle of the S-shaped PEG, a water molecule makes alternating hydrogen bonds to the ether oxygen atoms, in a similar configuration to that of a crown ether-bound lysine. Each satellite fragment is clamped between two arginine residues, R52 from the heavy chain and R29 from the light chain, and also interacts with several aromatic side chains. In contrast, the non-specifically bound PEG fragments in the 32D6-Fab crystal are located in the elbow region or at lattice contacts. The AUC data suggest that 3.3-Fab exists as a monomer in PEG-free solution but forms a dimer in the presence of PEG-550-MME, which is about the size of the S-shaped core PEG fragment. Conclusions The differing amino acids in 3.3 and 2B5 are not involved in PEG binding but engaged in dimer formation. In particular, the light-chain residue K53 of 2B5-Fab makes significant contacts with the other Fab in a dimer, whereas the corresponding N53 of 3.3-Fab does not. This difference in the protein-protein interaction between two Fab molecules in a dimer may explain the temperature dependence of 2B5 in PEG binding, as well as its inhibition by crown ether.

scholarly journals Conserved charged residues in the leucine-rich repeat domain of the Ran GTPase activating protein are required for Ran binding and GTPase activation

1999 ◽  
Vol 343 (3) ◽  
pp. 653-662 ◽  
Author(s):  
Jörg HABERLAND ◽  
Volker GERKE
Keyword(s):  
Nucleocytoplasmic Transport ◽  
Small Gtpases ◽  
Structural Basis ◽  
Ran Gtpase ◽  
Protein Protein Interaction ◽  
Charged Residues ◽  
Gtpase Activation ◽  
Gtpase Cycle ◽  
Leucine Rich Repeats ◽  
Lrr Domain

GTPase activating proteins (GAPs) for Ran, a Ras-related GTPase participating in nucleocytoplasmic transport, have been identified in different species ranging from yeast to man. All RanGAPs are characterized by a conserved domain consisting of eight leucine-rich repeats (LRRs) interrupted at two positions by so-called separating regions, the latter being unique for RanGAPs within the family of LRR proteins. The cytosolic RanGAP activity is essential for the Ran GTPase cycle which in turn provides directionality in nucleocytoplasmic transport, but the structural basis for the interaction between Ran and its GAP has not been elucidated. In order to gain a better understanding of this interaction we generated a number of mutant RanGAPs carrying amino acid substitutions in the LRR domain and analysed their complex formation with Ran as well as their ability to stimulate the intrinsic GTPase activity of the G protein. We show that conserved charged residues present in the separating regions of the LRR domain are indispensable for efficient Ran binding and GAP activity. These separating regions contain three conserved arginines which could possibly serve as catalytic residues similar to the arginine fingers identified in GAPs for other small GTPases. However, mutations in two of these arginines do not affect the GAP activity and replacement of the third conserved arginine (Arg91 in human RanGAP) severely interferes not only with GAP activity but also with Ran binding. This indicates that RanGAP-stimulated GTP hydrolysis on Ran does not involve a catalytic arginine residue but requires certain charged residues of the LRR domain of the GAP for mediating the protein-protein interaction.

scholarly journals Do antibodies recognize amino acid side chains of protein antigens independently of the carbon backbone?

1988 ◽  
Vol 167 (6) ◽  
pp. 1841-1848 ◽  
Author(s):  
V H Van Cleave ◽  
C W Naeve ◽  
D W Metzger
Keyword(s):  
Amino Acid ◽  
Peptide Sequence ◽  
Structural Basis ◽  
Side Chains ◽  
Protein Antigens ◽  
Carbon Backbone ◽  
Internal Image ◽  
Amino Acid Side Chains ◽  
V Region ◽  
Complementarity Determining Region

In an effort to understand the structural basis for antigen mimicry by internal image antibodies, we determined the variable (V) region sequences of two mouse mAbs that mimic the rabbit Ig a1 allotype. The results showed that while the mAb light chains did not contain any allotype-related residues, both heavy chain V regions contained within complementarity-determining region 2 an unusual sequence homologous to the nominal antigen but in opposite orientation with respect to the carbon backbone. The ability of the internal image reversed sequence to express an a1-like determinant was tested directly by producing synthetic peptides that corresponded to the presumed antigenic regions of rabbit Ig and the mAb internal images, respectively. Although the two peptides presented the homologous residues in opposite orientations, they both completely inhibited at similar concentrations the binding of rabbit Ig to anti-a1 antibody. Conservative substitutions in the peptide sequence identified a paired Thr and Glu as being critical for expression of the a1 epitope. These findings indicate that antibodies can recognize the molecular environments created by amino acid side chains independently from the orientation of the protein carbon backbone.

scholarly journals Crystal structure of yeast Sis1 peptide-binding fragment and Hsp70 Ssa1 C-terminal complex

2006 ◽  
Vol 398 (3) ◽  
pp. 353-360 ◽  
Author(s):  
Jingzhi Li ◽  
Yunkun Wu ◽  
Xinguo Qian ◽  
Bingdong Sha
Keyword(s):  
Crystal Structure ◽  
Close Contact ◽  
Critical Role ◽  
Peptide Binding ◽  
Structural Basis ◽  
Cellular Processes ◽  
Terminal Form ◽  
Charged Residues ◽  
Domain I

Heat shock protein (Hsp) 40 facilitates the critical role of Hsp70 in a number of cellular processes such as protein folding, assembly, degradation and translocation in vivo. Hsp40 and Hsp70 stay in close contact to achieve these diverse functions. The conserved C-terminal EEVD motif in Hsp70 has been shown to regulate Hsp40–Hsp70 interaction by an unknown mechanism. Here, we provide a structural basis for this regulation by determining the crystal structure of yeast Hsp40 Sis1 peptide-binding fragment complexed with the Hsp70 Ssa1 C-terminal. The Ssa1 extreme C-terminal eight residues, G634PTVEEVD641, form a β-strand with the domain I of Sis1 peptide-binding fragment. Surprisingly, the Ssa1 C-terminal binds Sis1 at the site where Sis1 interacts with the non-native polypeptides. The negatively charged residues within the EEVD motif in Ssa1 C-terminal form extensive charge–charge interactions with the positively charged residues in Sis1. The structure-based mutagenesis data support the structural observations.

scholarly journals Structural basis for amino acid exchange by a human heteromeric amino acid transporter

2020 ◽  
Vol 117 (35) ◽  
pp. 21281-21287 ◽  
Author(s):  
Di Wu ◽  
Tamara N. Grund ◽  
Sonja Welsch ◽  
Deryck J. Mills ◽  
Max Michel ◽  
...  
Keyword(s):  
Amino Acid ◽  
Heavy Chain ◽  
Light Chain ◽  
Basic Amino Acid ◽  
Binding Pocket ◽  
Structural Basis ◽  
Amino Acid Transporters ◽  
Wide Range ◽  
Functional Complex ◽  
Protein Components

Heteromeric amino acid transporters (HATs) comprise a group of membrane proteins that belong to the solute carrier (SLC) superfamily. They are formed by two different protein components: a light chain subunit from an SLC7 family member and a heavy chain subunit from the SLC3 family. The light chain constitutes the transport subunit whereas the heavy chain mediates trafficking to the plasma membrane and maturation of the functional complex. Mutation, malfunction, and dysregulation of HATs are associated with a wide range of pathologies or represent the direct cause of inherited and acquired disorders. Here we report the cryogenic electron microscopy structure of the neutral and basic amino acid transport complex (b[0,+]AT1-rBAT) which reveals a heterotetrameric protein assembly composed of two heavy and light chain subunits, respectively. The previously uncharacterized interaction between two HAT units is mediated via dimerization of the heavy chain subunits and does not include participation of the light chain subunits. The b(0,+)AT1 transporter adopts a LeuT fold and is captured in an inward-facing conformation. We identify an amino-acid–binding pocket that is formed by transmembrane helices 1, 6, and 10 and conserved among SLC7 transporters.

scholarly journals Synthetic peptides corresponding to third hypervariable region of human monoclonal IgM rheumatoid factor heavy chains define an immunodominant idiotype.

1985 ◽  
Vol 162 (2) ◽  
pp. 756-761 ◽  
Author(s):  
R D Goldfien ◽  
P P Chen ◽  
S Fong ◽  
D A Carson
Keyword(s):  
Rheumatoid Factor ◽  
Heavy Chain ◽  
Synthetic Peptides ◽  
Gene Segment ◽  
Hypervariable Region ◽  
Heavy Chains ◽  
The Third ◽  
Specific Induction ◽  
Antiidiotypic Antibodies ◽  
Complementarity Determining Regions

Synthetic peptides corresponding to eight individual heavy chain complementarity-determining regions (CDR) of three human monoclonal IgM anti-IgG (rheumatoid factor [RF]) paraproteins elicited rabbit antibodies with markedly different properties. All antisera recognized the immunizing peptide, and several reacted with the isolated IgM heavy chain on immunoblots. However, only the antisera against peptides representing the third CDR bound consistently and specifically to the intact IgM-RF molecule. These data indicate that the third CDR of human mu chains comprises an immunodominant idiotype, and suggest that the D gene segment may be especially important in creating idiotypic diversity. Synthetic peptides corresponding to the third heavy chain CDR of human paraproteins may be clinically useful for the specific induction of antiidiotypic antibodies.

scholarly journals Determination of the structural basis for selective binding of Epstein-Barr virus to human complement receptor type 2.

1991 ◽  
Vol 174 (6) ◽  
pp. 1299-1311 ◽  
Author(s):  
D R Martin ◽  
A Yuryev ◽  
K R Kalli ◽  
D T Fearon ◽  
J M Ahearn
Keyword(s):  
Epstein Barr Virus ◽  
Specific Binding ◽  
Glycosylation Site ◽  
Single Amino Acid ◽  
Structural Basis ◽  
Viral Receptor ◽  
Barr Virus ◽  
Preferential Binding ◽  
Epstein Barr ◽  
Species Specific

Epstein-Barr virus (EBV) is an oncogenic herpesvirus that selectively infects and immortalizes human B lymphocytes. One determinant of this narrow tropism is human CR2, the only viral receptor within the superfamily of proteins that contain short consensus repeats (SCRs). Human CR2 serves as a receptor for both C3dg and the gp350/220 glycoprotein of EBV, and binds the monoclonal antibody (mAb) OKB7, which blocks binding of both ligands to the receptor. In contrast, although murine CR2 is capable of binding human C3dg and this interaction can be blocked with the mAb 7G6, it does not bind OKB7 or EBV. We have determined the structural basis for absolute specificity of EBV for human CR2 through characterization of a panel of 24 human-murine chimeric receptors, all of which bind human C3dg. The results indicate that preferential binding of EBV to human CR2 is not due to unique amino acids that are capable of binding the virus, but reflects a distinct receptor conformation that can be achieved in murine CR2 with single amino acid substitutions in two discontinuous regions of the primary structure: replacement of proline at position 15 with the corresponding serine from human CR2, and elimination of a potential N-linked glycosylation site between SCR-1 and SCR-2. Furthermore, species-specific binding of EBV, OKB7, and 7G6 can all be manipulated through substitutions among residues 8-15, suggesting that this octapeptide is part of a structural determinant that is critical for binding of both viral and natural ligands to CR2.

Biochemical genetics of the mouse IgM system

1984 ◽  
Vol 62 (4) ◽  
pp. 217-224 ◽  
Author(s):  
Marc J. Shulman ◽  
Robert G. Hawley ◽  
Atsuo Ochi ◽  
W. O. T. Baczynsky ◽  
Catherine Collins ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Lines ◽  
Heavy Chain ◽  
Mutant Cell ◽  
Immunoglobulin M ◽  
Structural Basis ◽  
Deletion Mutants ◽  
Transfer System ◽  
Biochemical Genetics ◽  
Chain Synthesis

Using a mouse hybridoma system, we have developed methods of isolating a variety of mutant cell lines in which immunoglobulin function or synthesis is defective. The analysis of mutants defective in κ chain synthesis has defined a class of murine transposons. The deletion mutants produce immunoglobulin M (IgM) bearing μ heavy chain fragments and provide information on the requirements of IgM assembly and μ gene expression. We also describe a transfer system for the μ and κ genes which will be useful in analyzing the structural basis of IgM function.

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