Biomedical subjects

Merrill J Rowley

Publications and source records attributed to Merrill J Rowley.

17 recordsLinked to original sources

PubMed2006

Molecular characterization of a disease associated conformational epitope on GAD65 recognised by a human monoclonal antibody b96.11.

Autoantibodies to the 65kDa isoform of glutamate decarboxylase (GAD65) are associated with type I diabetes and recognise highly conformational epitope(s) that remain to be defined. The human recombinant Fab from mAb b96.11 inhibits binding of most GAD65 antibody positive sera from patients and its epitope has previously been localized to the middle region of GAD65. Recent studies indicate that b96.11 antibody specificity predicts the risk of developing type 1 diabetes in prediabetic individuals. We describe the use homology modelling, protein-protein docking simulations and biopanning of random peptide phage displayed libraries with b96.11 to predict contact amino acids on the interface of GAD65/Fab b96.11 complex. Further analysis by in vitro mutagenesis of GAD65 followed by radioimmunoprecipitation refined the amino acids contributing to the b96.11 epitope. Our studies show an interface characterized by a protruding antibody-combining site centered on the long heavy chain CDR3 loop of Fab b96.11 establishing interactions with the critical residue Phe(344) in the core of the epitope on GAD65, surrounded by charged sites within (375)RK(376) and (305)DER(307). The epitope requires residues from both middle and the C-terminal domains, and is the first precise definition of an epitope on GAD65. The nature of the b96.11 epitope leads to considerations of potential structural variations for differences in antigenicity between the isoforms GAD65 and GAD67. The study shows the utility of using a combination of in silico techniques and experimental data for molecular characterization and localization of conformational epitopes for which crystal structures are lacking.

Amino Acid Sequence↗

PubMed2006

Characterisation of an autoreactive conformational epitope on GAD65 recognised by the human monoclonal antibody b78 using a combination of phage display, in vitro mutagenesis and molecular modelling.

Autoantibodies to the diabetes autoantigen, the 65kDa isoform of glutamic acid decarboxylase (GAD65), react with conformational epitopes defined according to linear sequences but not according to structural information, or contact sites with the antibody paratope. To ascertain such information for an exemplary human monoclonal antibody (mAb) to GAD65, b78, we combined antibody screening of phage-displayed peptide libraries, alanine mutagenesis of selected motifs, homology modelling of the PLP and C-terminal regions of GAD65, and molecular dynamics to examine for structural effects of mutagenesis. By phage display, mAb b78 selected phagotopes containing acidic residues (D, E), hydrophobic residues (Y, F or W) and LRS that localised to a possible surface-exposed conformational epitope on the combined homology model. Alanine mutants of GAD65 based on deduced contact residues were examined for binding with b78 and control sera. Mutation of (524)SRL(526), (572)DF(573) and (498)KPQ(500) reduced reactivity of b78 with mutant GAD65 > 50%. Molecular dynamics indicated that mutation of (498)KPQ(500) caused structural changes that could account for effects of this mutation. Thus phage display in combination with molecular modelling identified contact residues within a highly conformational epitope for mAb b78 in the C-terminus of GAD65. These techniques should have broad applicability to definition of epitope structure.

Amino Acid Sequence↗

PubMed2005

Destructive effects of murine arthritogenic antibodies to type II collagen on cartilage explants in vitro.

Certain monoclonal antibodies (mAbs) to type II collagen (CII) induce arthritis in vivo after passive transfer and have adverse effects on chondrocyte cultures and inhibit self assembly of collagen fibrils in vitro. We have examined whether such mAbs have detrimental effects on pre-existing cartilage. Bovine cartilage explants were cultured over 21 days in the presence of two arthritogenic mAbs to CII (CIIC1 or M2139), a non-arthritogenic mAb to CII (CIIF4) or a control mAb (GAD6). Penetration of cartilage by mAb was determined by immunofluorescence on frozen sections and correlated with changes to the extracellular matrix and chondrocytes by morphometric analysis of sections stained with toluidine blue. The effects of mAbs on matrix components were examined by Fourier transform infrared microspectroscopy (FTIRM). A possible role of Fc-binding was investigated using F(ab)2 from CIIC1. All three mAbs to CII penetrated the cartilage explants and CIIC1 and M2139, but not CIIF4, had adverse effects that included proteoglycan loss correlating with mAb penetration, the later development in cultures of an abnormal superficial cellular layer, and an increased proportion of empty chondrons. FTIRM showed depletion and denaturation of CII at the explant surface in the presence of CIIC1 or M2139, which paralleled proteoglycan loss. The effects of F(ab)2 were greater than those of intact CIIC1. Our results indicate that mAbs to CII can adversely affect preformed cartilage, and that the specific epitope on CII recognised by the mAb determines both arthritogenicity in vivo and adverse effects in vitro. We conclude that antibodies to CII can have pathogenic effects that are independent of inflammatory mediators or Fc-binding.

Animals↗

PubMed2005

Amino acids critical for binding of autoantibody to an immunodominant conformational epitope of the pyruvate dehydrogenase complex subunit E2: identification by phage display and site-directed mutagenesis.

The E2 subunit of the mitochondrial multienzyme pyruvate dehydrogenase complex (PDC-E2) is the major autoantigen in the liver disease, primary biliary cirrhosis (PBC). An epitope region which has been localized to amino acids 91-227 is believed to include the residue K173 to which is attached the lipoyl cofactor. We investigated structural features of this epitope region by screening random peptide phage-displayed libraries and identified prevalent phagotopes that contained likely contact amino acids in separate regions of the linear sequence, H132M133, and F178, V180. These were confirmed by site-directed alanine mutagenesis singly or in combination of the HM and FV residues in wild-type (wt) PDC-E2, and by immunization of rabbits with phage that expressed peptides MHLNTPP or FVLPWRI. The lipoyl lysine K173 also was mutated. Reactivities of mutants and wild-type (wt) PDC-E2, compared by ELISA using 12 PBC sera, showed decremental reactivity of mutant versus wt PDC-E2 (normalized to 100%): wt PDC-E2 (100%)>>PDC-E2(F178A,V180A) (mean+/-S.D., 59+/-17%)>PDC-E2(M133A) (50+/-13%)>PDC-E2(H132A) (36+/-13%)>PDC-E2(H132A,M133A) (28+/-8%)>PDC-E2(H132A,M133A,F178V,M180A) (18+/-13%). Notably PDC-E2(K173A) retained full reactivity (93+/-21%). Rabbits immunized with phage peptides generated antibodies reactive with entire PDC-E2. Our data convincingly validate phage library technology for defining spatially disparate contact residues for conformational epitopes. Ensuing data could be generally applicable to search for occult extrinsic agents as initiators of autoimmunity.

Amino Acid Sequence↗

PubMed2005

Requirement of multiple phage displayed peptide libraries for optimal mapping of a conformational antibody epitope on CCR5.

In the absence of information from crystallography, conformational epitopes can often be discerned by antibody screening of phage displayed random peptide libraries. However the context in which the peptide is displayed, and the number of copies displayed in the library, can influence results and interpretations. Here, the monoclonal antibodies 3A9 specific for the transmembrane chemokine receptor CCR5, and CII-C1 specific for type II collagen, were used to screen multiple phage-displayed peptide libraries in which peptides were displayed in either the pIII or pVIII coat proteins. ELISA was used to test for reactivity and cross-inhibitory activity of isolated phage clones. Based on sequences of reactive phage inserts, epitope motifs were initially inferred from a molecular model of CCR5 and subsequently confirmed experimentally using mutagenesis to alanine. For each mAb, phage sequences from pIII biopannings were more diverse than from pVIII biopannings. Notably, sequences from either biopanning were cross-inhibitory despite a lack of linear sequence homology. For CCR5, residues 88H and 94W in the first loop of CCR5 were identified by pIII biopannings, and 7S9IYD11 at the N-terminus by pVIII biopannings. Thus conformational epitopes can be identified using phage display, but optimal mapping of complex epitopes can require the use of multiple peptide libraries.

Amino Acid Motifs↗

PubMed2005

Arthritogenic anti-type II collagen antibodies are pathogenic for cartilage-derived chondrocytes independent of inflammatory cells.

OBJECTIVE: Some monoclonal antibodies (mAb) to type II collagen (CII) are arthritogenic upon passive transfer to mice. We undertook this study to investigate whether such mAb are pathogenic in the absence of mediators of inflammation. METHODS: The arthritogenic mAb CIIC1 and M2139, and the nonarthritogenic mAb CIIF4, each reactive with a distinct and well-defined conformational epitope on CII, were compared with control mAb GAD6. Bovine chondrocytes were cultured with one of the mAb, and on days 3, 6, and 9, antibody binding by chondrocytes and newly synthesized extracellular matrix (ECM) was examined by immunofluorescence, morphologic effects were studied by electron microscopy, and synthesis of matrix components was determined by metabolic labeling with (3)H-proline for collagen and (35)S-sulfate for proteoglycans. RESULTS: All 3 mAb to CII bound to the matrix. CIIC1 and M2139 adversely affected the cultures, whereas CIIF4 did not. CIIC1 caused disorganization of CII fibrils in the ECM without affecting chondrocyte morphology, and increased matrix synthesis. M2139 caused thickening and aggregation of CII fibrils in the ECM and abnormal chondrocyte morphology but matrix synthesis was unaffected. CONCLUSION: The unique arthritogenic capacity of particular anti-CII mAb upon passive transfer could be explained by their adverse, albeit differing, effects in primary cultures of chondrocytes. Such effects occur independent of inflammation mediators and are related to the epitope specificity of the mAb. Interference with the structural integrity of CII could precede, and even initiate, the inflammatory expression of disease.

Animals↗

PubMed2005

Heritability of levels of autoantibodies using the method of plotting regression of offspring on midparent (ROMP).

The genetic control of the levels of autoantibodies has rarely been examined. We examined the heritability of autoantibodies to glutamic acid decarboxylase (GAD65) in type 1 diabetes, and to thyroglobulin (Tg) in chronic lymphocytic thyroiditis and thyrotoxicosis, using regression of offspring on midparent (ROMP) methods. Levels of autoantibodies in patients and their parents were significantly correlated in thyrotoxicosis (R2 = 0.569, p = 0.001), consistent with the reported Gm association, but not in chronic lymphocytic thyroiditis or type 1 diabetes. Extension of the procedure to other autoantibody disorders could be informative.

Adult↗

PubMed2004

Antibodies against the CB10 fragment of type II collagen in rheumatoid arthritis.

Antibodies against intact type II collagen (CII) are a feature of rheumatoid arthritis (RA) but have limited diagnostic value. Here we assess whether either of the two major cyanogen bromide fragments of CII, namely CB10 or CB11, are more sensitive substrates for the detection of antibodies in RA. Cleavage of bovine CII with cyanogen bromide yielded CB10 and CB11; these were purified by column chromatography for use in an enzyme-linked immunosorbent assay. Serum antibodies were measured in patients with RA, psoriatic arthritis (PsA), osteoarthritis (OA) and blood donors. Results were compared with those using intact CII. Antibodies against CB10 were found in as many as 88% of 96 patients with long-standing RA, but only 12% of 33 patients with PsA, 6% of 34 patients with OA and 3% of 93 control sera. Lower frequencies for these diseases were obtained on testing for antibodies against CB11: 50%, 6%, 21% and 2%, respectively. The sensitivity of detection in RA of antibodies against CB10 compared with antibodies against intact CII (88% versus 24%) was not at the expense of specificity, which remained high at 94%. The much higher frequency of antibodies against CB10 in RA than in other rheumatic diseases or control sera indicates that CB10 is clearly a more sensitive substrate than the intact collagen molecule and, combined with other assays (rheumatoid factor, anti-cyclic citrullinated peptide [anti-CCP]), might comprise a panel with a highly reliable predictive value. Moreover, our findings should encourage renewed interest in the role of collagen autoimmunity in the pathogenesis of RA.

Adolescent↗

PubMed2004

Measurement of antibodies to collagen II by inhibition of collagen fibril formation in vitro.

Antibodies to type II collagen (collagen II) are pathogenic in experimental collagen-induced arthritis (CIA) and possibly also in rheumatoid arthritis (RA). Hitherto, results of assays for anti-collagen II have proven to be inconsistent. We tested whether mouse monoclonal antibodies (mAbs) to collagen II inhibit the natural self-assembly of soluble triple-stranded collagen II monomers to form insoluble polymeric fibrils. A spectrophotometric assay of self-assembly was based on change in absorbance at 313 nm, observed over 0-60 min after neutralisation and warming of a solution of monomeric collagen II. Two mAbs to collagen II (CII-CI and M2.139) strongly inhibited self-assembly of collagen II but not collagen I, whereas another antibody, CII-F4, and an irrelevant control mAb did not. Notably, CII-CI and M2.139, but not CII-F4, induce arthritis on passive transfer to naïve mice. The arthritogenic effects of mAbs CII-CI and M2.139 in vivo, and inhibition of collagen II self-assembly in vitro, may be attributable to interference with critical epitopes at sites essential for the stabilisation of the mature polymeric collagen II fibril, and, hence, the integrity of the entire cartilage matrix. This assay for inhibition of self-assembly of collagen II could be developed for routine measurement of anti-collagen II in body fluids as a marker of early RA, and perhaps also to distinguish populations of antibodies to collagen II that either have or lack the capacity to perpetuate arthritis.

Animals↗

PubMed2004

Autoimmune epitopes: autoepitopes.

The identity of reactants for autoantibodies has been successively refined from whole cellular organelles (immunofluorescence), identified molecules (immunoblot; gene expression libraries), epitope regions (truncated cDNAs; peptide scanning) to contact residues, as described here. Most autoantibodies react with conformational epitopes, in which amino acids distant in the linear sequence come into contiguity by protein folding. Identification of contact sites with the antibody paratope requires particular technologies, crystallography, or antibody screening of phage-displayed random peptide libraries. The latter is illustrated by our studies on the autoepitope for anti-PDC-E2 (AMA) in primary biliary cirrhosis (PBC), anti-GAD65 in type 1 diabetes, and anti-C1 of type II collagen in collagen-induced arthritis. More precise definition of the structure of conformational autoepitopes could (a) clarify controversial aspects of autoimmunity including epitope mimicry, epitope spreading, and molecular spatial relationships between B and T cell autoepitopes, and (b) impact on novel diagnostic and therapeutic (vaccine) molecules.

Animals↗

PubMed2004

Two monoclonal antibodies to precisely the same epitope of type II collagen select non-crossreactive phage clones by phage display: implications for autoimmunity and molecular mimicry.

Two monoclonal antibodies (mAb) CB268 and CII-C1 to type II collagen (CII) react with precisely the same conformational epitope constituted by the residues ARGLT on the three chains of the CII triple helix. The antibodies share structural similarity, with most differences in the complementarity determining region 3 of the heavy chain (HCDR3). The fine reactivity of these mAbs was investigated by screening two nonameric phage-displayed random peptide libraries. For each mAb, there were phage clones (phagotopes) that reacted strongly by ELISA only with the selecting mAb, and inhibited binding to CII only for that mAb, not the alternate mAb. Nonetheless, a synthetic peptide RRLPFGSQM corresponding to an insert from a highly reactive CII-C1-selected phagotope, which was unreactive (and non-inhibitory) with CB268, inhibited the reactivity of CB268 with CII. Most phage-displayed peptides contained a motif in the first part of the molecule that consisted of two basic residues adjacent to at least one hydrophobic residue (e.g. RRL or LRR), but the second portion of the peptides differed for the two mAbs. We predict that conserved CDR sequences interact with the basic-basic-hydrophobic motif, whereas non-conserved amino acids in the binding sites (especially HCDR3) interact with unique peptide sequences and limit cross-reactivity. The observation that two mAbs can react identically with a single epitope on one antigen (CII), but show no cross-reactivity when tested against a second (phagotope) indicates that microorganisms could exhibit mimics capable of initiating autoimmunity without this being evident from conventional assays.

Amino Acid Motifs↗

PubMed2004

Phage display for epitope determination: a paradigm for identifying receptor-ligand interactions.

Antibodies that react with many different molecular species of protein and non-protein nature are widely studied in biology and have particular utilities, but the precise epitopes recognized are seldom well defined. The definition of epitopes by X-ray crystallography of the antigen-antibody complex, the gold standard procedure, has shown that most antibody epitopes are conformational and specified by interactions with topographic determinants on the surface of the antigenic molecule. Techniques available for the definition of such epitopes are limited. Phage display using either gene-specific libraries, or random peptide libraries, provides a powerful technique for an approach to epitope identification. The technique can identify amino acids on protein antigens that are critical for antibody binding and, further, the isolation of peptide motifs that are both structural and functional mimotopes of both protein and non-protein antigens. This review discusses techniques used to isolate such mimotopes, to confirm their specificity, and to characterize peptide epitopes. Moreover there are direct practical applications to deriving epitopes or mimotopes by sequence, notably the development of new diagnostic reagents, or therapeutic agonist or antagonist molecules. The techniques developed for mapping of antibody epitopes are applicable to probing the origins of autoimmune diseases and certain cancers by identifying "immunofootprints" of unknown initiating agents, as we discuss herein, and are directly applicable to examination of a wider range of receptor-ligand interactions.

Binding Sites↗

PubMed2004

An arthritogenic monoclonal antibody to type II collagen, CII-C1, impairs cartilage formation by cultured chondrocytes.

Antibodies to type II collagen (CII) cause articular damage in collagen-induced arthritis (CIA) in mice as judged by passive transfer to naive animals of mAb to CII. We tested the hypothesis that mAb degrade cartilage structure by reacting with functionally important regions of the collagen molecule by examining the effects of an arthritogenic mAb to CII, CII-C1, on cultured bovine chondrocytes at high density, at days 7 and 14. The effects were compared of CII-C1, an isotype-matched control mAb, or medium alone, on chondrocyte proliferation and viability, cell morphology, matrix structure by light and electron microscopy, and matrix synthesis by metabolic labelling with 3H-proline for collagen or 35SO4 for proteoglycans. Chondrocytes in culture remained viable, proliferated, and produced an extracellular matrix in which CII was the major collagen. The addition of CII-C1, but not a control mAb, increased the synthesis of CII and proteoglycan, and caused disorganization of the extracellular matrix and thin collagen fibrils ultrastructurally. Moreover, using a cell-free assay, CII-C1 inhibited the normal self-assembly of collagen fibrils from CII in solution. The finding that the mAb to CII, CII-C1 has striking degradative effects in vitro on cartilage synthesis suggests that antibodies to collagen perpetuate the chronic phase of CIA and that, in mice at least, such antibodies are an important component of pathogenesis.

Animals↗

PubMed2003

A diabetes-related epitope of GAD65: a major diabetes-related conformational epitope on GAD65.

The 65-kDa isoform of glutamic acid decarboxylase (GAD65) is a major autoantigen in type 1 diabetes, and most patients have serum antibodies reactive with conformational epitopes on the GAD65 molecule. The aims of this study were to prepare mutants of GAD65 to further localize the type 1 diabetes epitope in the region of the PEVKEK loop of GAD65 and to identify the particular amino acids within the epitope that are recognized by autoimmune diabetes sera.

Autoantibodies↗

PubMed2002

Enzymatic characterization of a recombinant isoform hybrid of glutamic acid decarboxylase (rGAD67/65) expressed in yeast.

BACKGROUND AND AIMS: Glutamic acid decarboxylase (GAD, EC 4.1.1.15) catalyses the conversion of glutamate to gamma-aminobutyric acid (GABA). The 65 kDa isoform, GAD65 is a potent autoantigen in type 1 diabetes, whereas GAD67 is not. A hybrid cDNA was created by fusing a human cDNA for amino acids 1-101 of GAD67 to a human cDNA for amino acids 96-585 of GAD65; the recombinant (r) protein was expressed in yeast and was shown to have equivalent immunoreactivity to mammalian brain GAD with diabetes sera. We here report on enzymatic and molecular properties of rGAD67/65. METHODS: Studies were performed on enzymatic activity of rGAD67/65 by production of 3H-GABA from 3H-glutamate, enzyme kinetics, binding to the enzyme cofactor pyridoxal phosphate (PLP), stability according to differences in pH, temperature and duration of storage, and antigenic reactivity with various GAD-specific antisera. RESULTS: The properties of rGAD67/65 were compared with published data for mammalian brain GAD (brackets). These included a specific enzyme activity of 22.7 (16.7) nKat, optimal pH for enzymatic activity 7.4 (6.8), K(m) of 1.3 (1.3) mM, efficient non-covalent binding to the cofactor PLP, and high autoantigenic potency. The stability of rGAD67/65 was optimal over 3 months at -80 degrees C, or in lyophilized form at -20 degrees C. CONCLUSIONS: Hybrid rGAD67/65 has enzymatic and other properties similar to those of the mixed isoforms of GAD in preparations from mammalian brain as described elsewhere, in addition to its previously described similar immunoreactivity.

Catalysis↗

PubMed2002

Autoantibodies to the transcriptional factor SOX13 in primary biliary cirrhosis compared with other diseases.

The molecule SOX13 was initially identified as an autoantigen (ICA12) in Type 1 diabetes. SOX13 is a member of the SOX family of transcriptional regulatory proteins that contain a high mobility group (HMG) motif with structural similarity to HMG proteins 1 and 2. Antibodies to HMG 1 and 2 occur in autoimmune diseases of the liver and in ulcerative colitis. We measured the occurrence and levels of anti-SOX13 by radioimmunoprecipitation in primary biliary cirrhosis (PBC) and other diseases, and compared frequencies with anti-HMG measured by ELISA. Anti-SOX13 was detected in 18% of patients with PBC, 13% with autoimmune hepatitis, 18% with Type 1 diabetes, at lower frequencies in other conditions including the multisystem autoimmune diseases, systemic lupus erythematosus and rheumatoid arthritis, and in 1% of normal sera. Anti-HMG1 and anti-HMG2 occurred at frequencies of 30% and 35% respectively in PBC. Serum levels of anti-SOX13 and anti-HMG correlated significantly for PBC although not for Type 1 diabetes. Anti-SOX13 in PBC may occur merely as an immune response to products of damage to parenchymal tissue, or may be illustrative of a general proclivity of transcriptional regulatory proteins to elicit autoimmune responses.

Antigens, Nuclear↗

PubMed2002

The PEVKEK region of the pyridoxal phosphate binding domain of GAD65 expresses a dominant B cell epitope for type 1 diabetes sera.

Molecular mimicry between the 65-kDa isoform of glutamic acid decarboxylase (GAD65) and the protein 2C (P2C) of Coxsackie B4 virus (CBV) may initiate human type 1 diabetes. GAD65 contains a motif that has a 6-amino acid identity with CBV-P2C (PEVKEK), whereas the weakly autoantigenic isoform, GAD67, contains PEVKTK. A human-derived monoclonal antibody (mAb) MICA3 reacts with a surface loop of GAD65 that includes PEVKEK, and mutagenic deletion of this loop was shown to reduce reactivity of GAD with the mAb by 70%. To establish that the PEVKEK motif on GAD65 contains a major epitope for diabetes sera and to identify the amino acids involved, mutants of nucleotides of GAD65 and GAD67 at sites in the PEVKEK motif were created and the expressed proteins used for radioimmunoprecipitation (RIP) tests with sera from patients with type 1 diabetes. A potent mouse mAb (GAD6) to GAD65, and a rabbit polyclonal antibody (AB108) to GAD67, were used to standardize the reactivity of the diabetes sera with the mutant molecules. Of 45 type 1 diabetes sera tested, 30 (67%) had an 80% or greater reduction of reactivity to GAD65(delta258-270) vs. intact GAD65. Various single-surface amino acids in the PEVKEK epitope region of GAD65 were mutated, but most molecules carrying these mutations reacted similarly to the parent molecule. However after point mutation of the equivalent motif of GAD67 (PEVKTK to PEVKEK), there was an increase in the reactivity of 12 of 49 (24%) type 1 diabetes sera tested; 7 of 8 sera reactive with GAD67 showed increased reactivity with GAD67(T273E), and 5 previously negative sera gained reactivity with GAD67(T273E). Thus, the PEVKEK motif on GAD65 contributes to serologic reactivity of type 1 diabetes sera. This favors the hypothesis that CBV infection causes type 1 diabetes by the process of viral mimicry with cross-reactivity to a critical epitope of GAD65.

Amino Acid Sequence↗