Identification of a putative globulin-specific protein processor by using anti-idiotypic antibodies
--Faith C. Belanger and Alan L. Kriz
The major storage protein of maize embryos is an Mr 63,000 vicilin-like globulin designated GLB1 which is encoded by the Glb1 gene. GLB1 is synthesized as a prepro-proprotein which undergoes extensive post-translational processing (Kriz and Schwartz, Plant Physiol. 82:1069, 1986). The final processing step, controlled by the unlinked gene Mep (Schwartz, MGG 174:233, 1979), is a proteolytic cleavage near the amino terminus of the polypeptide (Belanger and Kriz, Plant Physiol. 91:636, 1989). Embryos homozygous for the recessive mep allele accumulate the processing intermediate proGLB1 (formerly designated GLB1'). It is not known if Mep encodes the protease which cleaves proGLB1 to form GLB1 or for a factor which regulates activity or specificity of a protease.
In an attempt to identify the Mep gene product, antibodies to a synthetic 30 amino acid peptide, designed to span the Mep-controlled cleavage site, were raised in rabbits. These antibodies were in turn used to raise anti-idiotype antibodies which should recognize proteins which interact with the peptide sequence used as the initial antigen. Immunoblot analysis of embryo protein body extracts indicated that the anti-idiotype antibodies recognized an Mr 25,000 polypeptide present in Mep/Mep embryos but absent in mep/mep embryos. A screen of a maize embryo cDNA expression library with the anti-idiotype antibodies resulted in the isolation of three cross-hybridizing clones, the longest of which was designated pcPOG1 (for Processor Of Globulin) and selected for further analysis. Northern blot analysis indicated that transcripts corresponding to pcPOG1 are present in developing embryos and in leaves of 7 day-old seedlings but not in mature embryos, etiolated shoots, roots, or developing endosperm. Transcripts corresponding to pcPOG1 are present, however, in developing mep/mep embryos.
Nucleotide sequence analysis of pcPOG1 revealed an open reading frame of 354 amino acids which is in the correct reading frame for the cDNA library expression vector. The predicted molecular weight for the deduced protein is 37,084 daltons. The deduced protein sequence contains several potential membrane-spanning domains and a consensus sequence for a flavin binding site. An extensive search of databases with either the pcPOG1 nucleotide or deduced protein sequence was unsuccessful in identifying sequences which exhibit significant degrees of homology. To obtain full-length clones corresponding to pcPOG1, the cDNA library was re-screened with a radiolabelled fragment from the 5' region of the original clone. Sequence analysis of this clone is underway.
Southern blot analysis of maize DNA indicates that pcPOG1 sequences represent a small gene family. The Mep gene is located on the long arm of chromosome 5 (near Pr), and localization of pcPOG1 to this region would strongly suggest that pcPOG1 corresponds to Mep . Two loci have been mapped by Paul Sisco (NC State, Raleigh) to chromosomes 6 and 3. A third locus remains to be localized.
We are currently attempting to determine the function of the protein encoded by pcPOG1. For this purpose, the pcPOG1 protein has been expressed in E. coli by using the pMAL-c vector (New England Biolabs) for subsequent purification and production of additional antibodies. We are also planning in vitro transcription and translation experiments to determine if the pcPOG1 protein is capable of processing proGLB1 to GLB1.
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