The Glb1-V allele encodes an arginine-rich, basic seed globulin

--Alan L. Kriz, Faith C. Belanger and Cheryl A. Green

The maize Glb1 gene, which encodes a vicilin-like embryo storage protein, is highly polymorphic, and several naturally occurring alleles have been described. The most common alleles, referred to as size alleles, encode mature polypeptides ranging in molecular mass from about Mr 60,000 to Mr 70,000, as determined by SDS-PAGE. We recently reported the nucleotide sequences of the Glb1-L (Large protein) and Glb1-S (Small protein) alleles (Genetics 129:863, 1991) and determined that the size difference between the two proteins encoded by these alleles is due to a 12 amino acid duplication in the GLB1-L protein relative to the GLB1-S protein. To further investigate the nature of allelic variation of Glb1, we cloned and sequenced the Glb1-V allele, the protein product of which has been reported by Osterman (Biochem. Genet. 26:463, 1988) to differ from the L and S proteins in the manner of protein processing. The sequence of Glb1-V is very similar to that of the other Glb1 alleles, except that deletion of a single residue in the last (fifth) exon results in a frameshift mutation which dramatically alters the characteristics of the protein, as determined by deduction of the amino acid sequence from the nucleotide sequence. The most notable difference between the GLB1-V protein and the other GLB1 proteins is an effective substitution of several glutamic acid residues in the carboxy-terminal third of the polypeptide with arginine residues, due to the change in reading frame. The net result of these acidic to basic amino acid substitutions is a drastic change in the isoelectric point of the protein from about 6.5 to 12.3. Some characteristics of the different allelic forms of mature Glb1 proteins, as deduced from nucleotide sequence information, are as follows:
mol. wt. 56,300 55,100 53,500
pI 6.2 6.8 12.3
# arg residues 61 59 83
# basic aa, total 100 98 115
# acidic aa, total 88 82 37

It is not apparent from the primary sequence of the GLB1-V protein how the processing of this polypeptide would differ from that of other GLB1 proteins. It is likely that the sequence changes introduced by the frameshift mutation alter the structure of the protein in such a manner as to result in altered processing. Analysis of GLB1-V protein synthesis may provide insight to the nature of globulin processing in maize embryos.

It is also interesting to note that the introduction of a large number of basic amino acid residues does not affect accumulation of the GLB1-V protein in maize embryos. One of the objectives of our laboratory is to use modified versions of the Glb1 gene, engineered to contain a large number of lysine codons, as a means of enhancing the nutritional quality of maize grain protein. Mother Maize has shown us that the seed is capable of tolerating a Glb1-encoded protein with a large number of basic arginine residues, and it is likely that modified polypeptides, containing large numbers of basic lysine residues rather than arginines, will effectively be accumulated in the seed as well.

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