Proteinase inhibitor from ginkgo seeds is a member of the plant nonspecific lipid transfer protein gene family.
A 9-kD proteinase inhibitor was isolated from the seeds of ginkgo (Ginkgo biloba) and purified to homogeneity. This protein was revealed to partial-noncompetitively inhibit the aspartic acid proteinase pepsin and the cysteine proteinase papain (inhibition constant = 10(-5)-10(-4) m). The cDNA of the inhibitor was revealed to contain a 357-bp open reading frame encoding a 119-amino acid protein with a potential signal peptide (27 residues), indicating that this protein is synthesized as a preprotein and secreted outside the cells. Semiquantitative reverse transcription-polymerase chain reaction revealed that this gene expresses only in seeds, not in stems, leaves, and roots, suggesting that the protein is involved in seed development and/or germination. The inhibitor showed about 40% sequence homology with type-I nonspecific lipid transfer protein (nsLTP1) from other plant species. Actually, this inhibitor exerted both lipid transfer activity and lipid-binding activity, while the protein did not show any antifungal and antibacterial activities. Furthermore, the site-directed mutagenesis study using a recombinant ginkgo nsLTP1 revealed that proline (Pro)-79 and phenylalanine-80 are important on phospholipid transfer activity and that Pro-79 and isoleucine-82 are essential for the binding activity toward cis-unsaturated fatty acids. On the other hand, the alpha-helical content of P79A and F80A mutants was significantly lower than that of the wild-type protein. It was noteworthy that the papain-inhibitory activity of P79A and F80A mutants was elevated twice as much as that of the wild-type protein. In summary, we concluded that Pro-79 plays a critical role in both the lipid transfer and binding activities of ginkgo nsLTP1.
Sawano
Yoriko
Y
Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, University of Tokyo, Bunkyo-ku, Tokyo 113-8657, Japan.
Hatano
Ken-ichi
K
Miyakawa
Takuya
T
Komagata
Hideki
H
Miyauchi
Yumiko
Y
Yamazaki
Hiroshi
H
Tanokura
Masaru
M
D000595:Amino Acid Sequence; D052179:Antigens, Plant / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D001483:Base Sequence; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D002942:Circular Dichroism; D003001:Cloning, Molecular; D017931:DNA Primers; D018076:DNA, Complementary; D004591:Electrophoresis, Polyacrylamide Gel; D020441:Ginkgo biloba / Q000196:embryology*; D007700:Kinetics; D008969:Molecular Sequence Data; D010940:Plant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D011480:Protease Inhibitors / Q000737:chemistry / Q000302:isolation & purification* / Q000494:pharmacology; D012639:Seeds / Q000737:chemistry*; D017386:Sequence Homology, Amino Acid
D052179:Antigens, Plant; D002352:Carrier Proteins; D017931:DNA Primers; D018076:DNA, Complementary; D010940:Plant Proteins; D011480:Protease Inhibitors; C511920:lipid transfer proteins, plant