Inhibitory Serpins from Wheat Grain with Reactive Centers Resembling Glutamine-rich Repeats of Prolamin Storage Proteins

Abstract

Genes encoding proteins of the serpin superfamily are widespread in the plant kingdom, but the properties of very few plant serpins have been studied, and physiological functions have not been elucidated. Six distinct serpins have been identified in grains of hexaploid bread wheat (Triticum aestivum L.) by partial purification and amino acid sequencing. The reactive centers of all but one of the serpins resemble the glutamine-rich repetitive sequences in prolamin storage proteins of wheat grain. Five of the serpins, classified into two protein Z subfamilies, WSZ1 and WSZ2, have been cloned, expressed in Escherichia coli, and purified. Inhibitory specificity toward 17 proteinases of mammalian, plant, and microbial origin was studied. All five serpins were suicide substrate inhibitors of chymotrypsin and cathepsin G. WSZ1a and WSZ1b inhibited at the unusual reactive center P 1 -P 1 Gln-Gln, and WSZ2b at P 2 -P 1 LeuArg-one of two overlapping reactive centers. WSZ1c with P 1 -P 1 Leu-Gln was the fastest inhibitor of chymotrypsin (k a ‫؍‬ 1 The serpins constitute a superfamily of versatile proteins participating in the regulation of complex proteolytic systems (1, 2). Most serpins are serine proteinase inhibitors of chymotrypsin-like enzymes, but a few have been found to inhibit cysteine proteinases (3), and some are non-inhibitory. Many functionally distinct serpins have been identified in higher eukaryotes, some in viruses, but none in yeast or bacteria (4, 5).Mammalian serpins have been shown to participate in a growing number of extra-and intracellular physiological processes, including blood coagulation, complement activation, remodeling of the extracellular matrix, and hormone transport.The only plant serpin characterized in detail with respect to inhibitory specificity is recombinant barley (Hordeum vulgare) serpin rBSZx 1 (6 -8), but BSZx (GenBank™ accession number X97636) has not been detected in the plant. In addition, two serpins from barley grain, BSZ4 (7, 9 -11) and BSZ7 (GenBank™ accession number CAA64599) (12, 13), and one from wheat (Triticum aestivum) grain (7, 14, 15) have been studied. Despite the high concentration of these inhibitors in the grains of monocot cereals (up to 4% total protein), the physiological functions of plant serpins remain unknown. Increasing numbers of putative serpin genes and serpin mRNAs have been identified from other monocot plants, including rice and wild oats, from eudicot plants, including tomato, cotton, and the model plant Arabidopsis thaliana, and from the non-vascular plant Physcomitrella patens (EMBL/GenBank), suggesting that serpins are widespread in the plant kingdom.Inhibitory serpins are metastable proteins (Ͼ40 kDa) employing a unique "suicide substrate" mechanism of irreversible inhibition very different from the reversible "standard mechanism" used by other proteinase inhibitors (Ͻ25 kDa) (1). The serpin in its native, active conformation has a flexible reactive center loop (RCL) located near the C terminus and protruding from the main body of the protein...

Miropin, a Novel Bacterial Serpin from the Periodontopathogen Tannerella forsythia, Inhibits a Broad Range of Proteases by Using Different Peptide Bonds within the Reactive Center Loop

“…This is a very unusual feature, as all other inhibitory serpins characterized to date, with few exceptions (41)(42)(43)(44), use a single polypeptide bond (P1-P1Ј) to trap a protease in a covalent complex. Of note, the RCL in miropin has the same length as the vast majority of eukaryotic and prokaryotic serpins (Figs.…”

Arabidopsis AtSerpin1, Crystal Structure and in Vivo Interaction with Its Target Protease RESPONSIVE TO DESICCATION-21 (RD21)

“…Serpins from cereal grains are irreversible inhibitors of serine proteases with distinct inhibitory specificity (10,11). The majority of inhibitory serpins from wheat and rye grain contain motifs within the RCL that resemble the glutamine-rich repeats of grain storage proteins, suggesting a function in the protection of storage protein degradation by exogenous proteases (12,13). In addition, the differential expression of serpins in barley grain suggested a function in seed survival within the herbivore digestive tract (14).…”

A Review: Biological and Technological Functions of Barley Seed Pathogenesis-Related Proteins (PRs)

“…The high content of lysine and gene expression in serpins regulated by the "high-Lys" alleles lys1 and lys3a 30 , as well as the pattern of accumulation and relative abundance during grain filling 90 , suggested their role as storage proteins in plants. Functions in defense have been suggested for cereal seed serpins 124,125,246,281 . Serpins are inactivated by low pH and thus not able to act as in vivo inhibitors of digestive proteinases in animals with acidic stomachs, but may protect storage proteins from insects' digestive serine proteinases of the chymotrypsin family in neutral or alkaline gut pH, or promote seed dispersal by seed-eating birds.…”