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1.
Biochemistry ; 59(23): 2171-2181, 2020 06 16.
Article in English | MEDLINE | ID: mdl-32459958

ABSTRACT

Interleukin-31 (IL-31) is a major protein involved in severe inflammatory skin disorders. Its signaling pathway is mediated through two type I cytokine receptors, IL-31RA (also known as the gp130-like receptor) and the oncostatin M receptor (OSMR). Understanding molecular details in these interactions would be helpful for developing antagonist anti-IL-31 monoclonal antibodies (mAbs) as potential therapies. Previous studies suggest that human IL-31 binds to IL-31RA and then recruits OSMR to form a ternary complex. In this model, OSMR cannot interact with IL-31 in the absence of IL-31RA. In this work, we show that feline IL-31 (fIL-31) binds independently with feline OSMR using surface plasmon resonance, an enzyme-linked immunosorbent assay, and yeast surface display. Moreover, competition experiments suggest that OSMR shares a partially overlapping epitope with IL-31RA. We then used deep mutational scanning to map the binding sites of both receptors on fIL-31. In agreement with previous studies of the human homologue, the binding site for IL31-RA contains fIL-31 positions E20 and K82, while the binding site for OSMR comprises the "PADNFERK" motif (P103-K110) and position G38. However, our results also revealed a new overlapping site, composed of positions R69, R72, P73, D76, D81, and E97, between both receptors that we called the "shared site". The conformational epitope of an anti-feline IL-31 mAb that inhibits both OSMR and IL-31RA also mapped to this shared site. Combined, our results show that fIL-31 binds IL-31RA and OSMR independently through a partially shared epitope. These results suggest reexamination of the putative canonical mechanisms for IL-31 signaling in higher animals.


Subject(s)
Epitopes/metabolism , Interleukins/metabolism , Oncostatin M Receptor beta Subunit/metabolism , Receptors, Interleukin/metabolism , Animals , Cats , Epitopes/chemistry , Humans , Interleukins/chemistry , Models, Molecular , Oncostatin M Receptor beta Subunit/chemistry , Receptors, Interleukin/chemistry
2.
Mol Immunol ; 59(1): 71-8, 2014 May.
Article in English | MEDLINE | ID: mdl-24509215

ABSTRACT

Complementarity determining regions (CDR) are responsible for binding antigen and provide substantial diversity to the antibody repertoire, with VH CDR3 of the immunoglobulin variable heavy (VH) domain playing a dominant role. In this study, we examined 1200 unique canine VH and 500 unique variable light (VL) sequences of large and small canine breeds derived from peripheral B cells. Unlike the human and murine repertoire, the canine repertoire is heavily dominated by the Canis lupus familiaris IGHV1 subgroup, evolutionarily closest to the human IGHV3 subgroup. Our studies clearly show that the productive canine repertoire of all analyzed breeds shows similarities to both human and mouse; however, there are distinct differences in terms of VH CDR3 length and amino acid paratope composition. In comparison with the human and murine antibody repertoire, canine VH CDR3 regions are shorter in length than the human counterparts, but longer than the murine VH CDR3. Similar to corresponding human and mouse VH CDR3, the amino acids at the base of the VH CDR3 loop are strictly conserved. For identical CDR positions, there were significant changes in chemical paratope composition. Similar to human and mouse repertoires, the neutral amino acids tyrosine, glycine and serine dominate the canine VH CDR3 interval (comprising 35%) although the interval is nonetheless relatively depleted of tyrosine when compared to human and mouse. Furthermore, canine VH CDR3 displays an overrepresentation of the neutral amino acid threonine and the negatively charged aspartic acid while proline content is similar to that in the human repertoire. In general, the canine repertoire shows a bias towards small, negatively charged amino acids. Overall, this analysis suggests that functional canine therapeutic antibodies can be obtained from human and mouse sequences by methods of speciation and affinity maturation.


Subject(s)
Complementarity Determining Regions/genetics , Gene Expression , Immunoglobulin Heavy Chains/genetics , Immunoglobulin Variable Region/genetics , Animals , Antibody Diversity/genetics , B-Lymphocytes/metabolism , Breeding , Dogs , Humans , Immunoglobulin G/genetics , Immunoglobulin M/genetics , Mice
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