Characterization of an Antiserum against Feather Keratins of the Chick: Its Crossreaction with a Lens Protein, δ‐crystallin

Abstract

We prepared an antiserum against a fraction of solubilized keratins extracted from down feathers of newly hatched chicks. The specificity of the antiserum was tested by double immunodiffusion, immunofluorescent staining, and immunoblotting after sodium dodecylsulfatepolyacrylamide gel electrophoresis. All the bands except "Fast protein" reacted with the antiserum, suggesting the presence of a common antigenicity through various polypeptides in solubilized feather keratins. Delta-crystallin, which is a lens specific protein, also reacted with the antiserum. The presence of a common antigenicity between d-crystallin and feather and scale keratins was confirmed by affinity-purification of the antiserum, and its significance is discussed.The avian feather shows distinct X-ray diffraction pattern from mammalian skin or hair and its constituent proteins, i.e. feather keratins are also distinct from mammalian keratins by their low molecular weight (see review, I). The molecular weight of feather keratins are estimated to be about 1 1 K, while those of mammalian keratins range from 40 K to 65 K (2). Since feather keratins are insoluble in aqueous buffer, reduction and S-carboxymethylation procedure have been adopted to solubilize them and make them applicable to biochemical analyses. Polyacrylamide gel electrophoresis at both acidic and basic pH show that S-carboxymethylated (SCM) feather keratins can be separated to several bands, suggesting the total number of homologous polypeptides to be at least 35 (3). This number coincides well with the estimated number of keratin mRNA in the embryonic chick feather (4). Partial amino acid sequence analysis also confirmed the microheterogeneity of keratin polypeptides (5).We previously reported that limb bud cells of 4-day-old chick embryo can synthesize a lens specific protein, 6-crystallin, when cultured as aggregates, and that those cells containing &crystallin are derived from both the ectoderm and the mesoderm of the limb bud (6). For the further analysis of this phenomenon, a probe for the normal differentiation of limb bud cells was needed. We selected feather keratins, as they are the terminal differentiative products of the ectodermal cells of the limb bud, and raised an antiserum against some of the feather keratin polypeptides. The specificity of the antiserum was checked by the double immunodiffusion test, the indirect immunofluorescent staining and the immunoblotting test. Unexpectedly, the antiserum also reacted with 6-crystallin. Delta-crystallin is not contained in feather at all and further purification of the antiserum showed that there is a group of antibodies * Present address:

Reliability of delta-crystallin as a marker for studies of chick lens induction

“…Second, there is a general concern that polyclonal antibodies sometimes cross-react with other proteins. This seems to be a potential problem for our studies because of a report of binding of antibodies to feather keratins to the deltacrystallin protein [18] and because delta-crystallin antibodies can bind to feather keratins (R.M. Grainger, personal communication).…”