Ginkgo 11S seed storage protein family mRNA: unusual Asn-Asn linkage as post-translational cleavage site

Abstract

By reducing the amount of ginkgo water-soluble polysaccharides, which occupy about 35% of the wet seed mass and interfere with the extraction of RNA, cDNA-quality mRNA was obtained from developing seeds of Ginkgo biloba. Based on the NH2-terminal 17-amino acid sequence and an internal 12-amino acid sequence derived from the basic subunit of ginnacin, 11S-seed storage protein family of ginkgo, two degenerate oligonucleotide primers were synthesized and used for polymerase chain reaction (PCR). The resulting PCR product was used for screening the above endosperm cDNA library, and a plaque carrying the 1614 bp cDNA insert, which contained the entire coding region for a precursor of ginnacin was isolated. This is the first reported cloning of cDNA from ginkgo seeds. The deduced primary sequence is composed of a signal peptide segment (25 amino acid residues) and an acidic subunit (248 residues) followed by a basic subunit (187 residues). It was also found that the post-translational cleavage site in the ginnacin precursor is the Asn-Asn rather than the Asn-Gly bond found in a variety of the major subunit precursors in 11S seed protein family known to date. We showed that a purified soybean extract and an extract of ginkgo seeds can specifically hydrolyze -Asn248-Asn249- but not -Asn249-Val250-, in the heptapeptide Gly-Asn248-Asn-Val-Glu-Glu-Leu that corresponds to the ginnacin cleavage region.

Proteinase Inhibitor from Ginkgo Seeds Is a Member of the Plant Nonspecific Lipid Transfer Protein Gene Family

“…Actually, ginkgo seeds contain several storage proteins such as a 30-kD storage glycoprotein (Kimura et al, 1999), the 11S seed storage proteins ginnacin (Arahira and Fukazawa, 1994) and legumin (Jensen and Berthold, 1989), and a-mannosidase (Woo et al, 2004). However, no proteinase activity has been detected in ginkgo seeds (data not shown); therefore, such endogenous proteinases might be expressed when the plant requires them, such as during seed germination.…”

Genomic organization of glycinin genes in soybean

“…That the conserved posttranslational cleavage site is NV rather than NG has precedent. Aberrant cleavage sites have been described previously in subunits from pea (March et al 1988), gingko (Arahira and Fukazawa 1994) and pine (Allona et al 1992). Deviations from the NG cleavage site usually present among the 11S subunits in angiosperms are widespread in the Taxodiaceae, Cupressaceae and Taxaceae (Häger and Fischer 1999).…”

Legumin encoding sequences from the Redwood family (Taxodiaceae) reveal precursors lacking the conserved Asn‐Gly processing site

“…the Asn-Asn bond of the peptide GNNVEEL is specifically cleaved by the enzyme [9]. The peptide corresponds to the processing region of a legumin precursor from Ginkgo, and the cleavage observed is in accordance with the in vivo processing of this precursor [9,13,23]. The specifity of a soybean maturation protease, different from that refered above, has been investigated using octapeptides or mutated prolegumins with altered amino acids at or adjacent to Asn-Gly bonds.…”

“…The proteases involved in legumin precursor processing actually do not show pronounced specifity for Asn-Gly cleavage [7][8][9][10]; this has been concluded from in vitro studies using artificial substrates. However, little attention has been drawn to precursors found to have divergent processing sites.…”