Corn kernel cysteine proteinase inhibitor as a novel cystatin superfamily member of plant origin. Molecular cloning and expression studies.

A full-length cDNA clone for a cysteine proteinase inhibitor (cystatin) was isolated from a lambda gt10 cDNA library of immature corn kernels by screening with a mixture of cDNA inserts for oryzacystatins I and II. The cDNA clone spans 960 base pairs, encoding a 135-amino-acid protein containing a signal peptide fragment. The protein, named corn cystatin I, is considered to be a member of the cystatin superfamily, since it contains the commonly conserved Gln-Val-Val-Ala-Gly region that exists in most known cystatins as a probable binding site and is significantly similar to other cystatins in its overall amino acid sequence. Corn cystatin I expressed in Escherichia coli showed a strong papain-inhibitory activity. Northern blot analysis showed that the amount of mRNA for corn cystatin I reaches a maximum 2 weeks after flowering and then decreases gradually.     

The platelet collagen receptor glycoprotein VI is a member of the immunoglobulin superfamily closely related to FcalphaR and the natural killer receptors.

We have cloned the platelet collagen receptor glycoprotein (GP) VI from a human bone marrow cDNA library using rapid amplification of cDNA ends with platelet mRNA to complete the 5' end sequence. GPVI was isolated from platelets using affinity chromatography on the snake C-type lectin, convulxin, as a critical step. Internal peptide sequences were obtained, and degenerate primers were designed to amplify a fragment of the GPVI cDNA, which was then used as a probe to screen the library. Purified GPVI, as well as Fab fragments of polyclonal antibodies made against the receptor, inhibited collagen-induced platelet aggregation. The GPVI receptor cDNA has an open reading frame of 1017 base pairs coding for a protein of 339 amino acids including a putative 23-amino acid signal sequence and a 19-amino acid transmembrane domain between residues 247 and 265. GPVI belongs to the immunoglobulin superfamily, and its sequence is closely related to FcalphaR and to the natural killer receptors. Its extracellular chain has two Ig-C2-like domains formed by disulfide bridges. An arginine residue is found in position 3 of the transmembrane portion, which should permit association with Fcgamma and its immunoreceptor tyrosine-based activation motif via a salt bridge. With 51 amino acids, the cytoplasmic tail is relatively long and shows little homology to the C-terminal part of the other family members. The ability of the cloned GPVI cDNA to code for a functional platelet collagen receptor was demonstrated in the megakaryocytic cell line Dami. Dami cells transfected with GPVI cDNA mobilized intracellular Ca(2+) in response to collagen, unlike the nontransfected or mock transfected Dami cells, which do not respond to collagen.     

Characterization and amino acid sequence of a new acidic cysteine proteinase inhibitor (cystatin SA) structurally closely related to cystatin S, from human whole saliva

A cysteine proteinase inhibitor (designated as cystatin SA) was isolated from human whole saliva by procedures including chromatography on DE 32 and DEAE-Sepharose CL-6B. The amino acid sequence determined by conventional methods showed sequence homology of 90 and 87% as compared with the sequences of cystatin S and cystatin SN, respectively, both of which are salivary inhibitors characterized previously. The new inhibitor consisted of 117 residues and had a pI value of 4.3. Cystatin SA inhibited ficin and papain more strongly than cystatin S or cystatin SN did. It also exhibited inhibitory activity toward dipeptidyl peptidase I but the activity was much weaker than those toward ficin and papain.     

Histidine-rich glycoprotein is evolutionarily related to the cystatin superfamily. Presence of two cystatin domains in the N-terminal region

A new member of the cystatin superfamily is introduced. Human plasma histidine-rich glycoprotein (HRG) was found to contain 2 cystatin-like sequences in tandem in the N-terminal region. Domain 1 (residues 1-112) was most homologous to domain 1 of the heavy chain of human kininogen and domain 2 (residues 113-225) was most homologous to human cystatin S as well as other cystatins and domain 3 of the heavy chain of kininogen, suggesting that the cystatin domains of HRG may represent a hitherto unknown binary form (or intermediate molecule) composed of 2 cystatin domains, and evolutionarily intermediate between the cystatin and the kininogen families.     

Characterization of a new cysteine proteinase inhibitor of human saliva, cystatin SN, which is immunologically related to cystatin S

A new cysteine proteinase inhibitor, cystatin SN, was purified from human whole saliva by chromatography with DE32, Sephacryl S200, and CM-Sepharose CL6B. Cystatin SN is immunologically related to cystatin S and both inhibitors have a similar molecular mass of about 13 kDa. The new inhibitor, however, was clearly distinguished from cystatin S by its much higher pI value. These inhibitors showed similar inhibitory activity for ficin, but cystatin SN was a much better inhibitor for papain and dipeptidyl peptidase I. The amino acid sequence of cystatin SN deduced in the light of the known structure of cystatin S indicates that they have 10 different amino acid residues in the sequence comprising in total 113 residues.     

Molecular characterization of a dsRNA mycovirus, Fusarium graminearum virus-DK21, which is phylogenetically related to hypoviruses but has a genome organization and gene expression strategy resembling those of plant potex-like viruses

Fusarium graminearum causes a serious scab disease of small grains in Korea. The nucleotide sequence of the genomic RNA of a double-stranded RNA (dsRNA) virus, Fusarium graminearum virus-DK21 (FgV-DK21), from F. graminearum strain DK21, which is associated with hypovirulence in F. graminearum, was determined and compared to the genome sequences of other mycoviruses, including Cryponectria hypoviruses. The FgV-DK21 dsRNA consists of 6,624 nucleotides, excluding the 3'-terminal poly(A) tail. The viral genome has 53- and 46-nucleotide 5' and 3' untranslated regions (UTRs), respectively, and five putative open reading frames. A phylogenetic analysis of the deduced amino acid sequence of ORF1, which encodes a putative RNA-dependent RNA polymerase, and those of other mycoviruses revealed that this organism forms a distinct virus clade with other hypoviruses, and is more distantly related to other mycoviruses (3.8 to 24.0% identity). However, pairwise sequence comparisons of the nucleotide and deduced amino acid sequences of ORFs 2 through 5 revealed no close relationships to other protein sequences currently available in GenBank. Analyses of RNA accumulation by Northern blot and primer extension indicated that these putative gene products are expressed from at least two different subgenomic RNAs (sgRNAs), in contrast to the cases in other hypoviruses. This study suggests the existence of a new, as yet unassigned, genus of mycoviruses that exhibits a potex-like genome organization and sgRNA accumulation.     

Rosmarinic acid synthase is a new member of the superfamily of BAHD acyltransferases

Purification of rosmarinic acid synthase (hydroxycinnamoyl-CoA:hydroxyphenyllactate hydroxycinnamoyltransferase) from suspension cells of Coleus blumei Benth. (Lamiaceae) by fractionated ammonium sulphate precipitation, hydrophobic interaction chromatography and two affinity chromatography steps led to the identification of peptide sequences, which enabled a PCR-based approach to isolate the full-length cDNA encoding this enzyme. The open reading frame of the cDNA had a length of 1290 base pairs encoding a protein of 430 amino acid residues with a molecular mass of 47,932 Da with typical characteristics of an acyltransferase of the BAHD superfamily. The cDNA was heterologously expressed in Escherichia coli. The enzyme displayed the activity of rosmarinic acid synthase using 4-coumaroyl- and caffeoyl-coenzyme A and 4-hydroxyphenyllactate as well as 3.4-dihydroxyphenyllactate as substrates. Shikimic acid and quinic acid were not able to serve as hydroxycinnamoyl acceptors. This therefore is the first report of the cDNA-cloning of a rosmarinic acid synthase.     

Calmodulin-binding protein BP-10, a probable new member of plant nonspecific lipid transfer protein superfamily.

CaMBP-10 is a novel plant endogenous calmodulin-binding protein with important physiological functions. The partial cDNA sequence of this protein was cloned using RT-PCR. The deduced peptide (designated PCBP10) is composed of 74 amino acid residues containing a basic amphiphilic alpha-helix typical for calmodulin-binding proteins. PCBP10 shows very high amino acid sequence homology with plant nonspecific lipid-transfer proteins (nsLTPs). Sequence analysis also reveals that PCBP10 has similar amino acid composition to plant nsLTPs, and seven of the eight conserved cysteine residues are found in PCBP10. Furthermore, the secondary structure features of PCBP10 are very similar to those of plant nsLTPs. In addition, there are striking resemblances between CaMBP-10 and plant nsLTPs in their biochemical and physical properties. Our results suggest that CaMBP-10 is a novel member of the plant and nsLTP gene family, and the Ca(2+)/CaM regulative system may also play roles in lipid metabolism, defense reactions, and the adaptation of plants to natural environment.     

Alpha-amino-beta-carboxymuconic-epsilon-semialdehyde decarboxylase (ACMSD) is a new member of the amidohydrolase superfamily

The enzymatic activity of Pseudomonas fluorescens alpha-amino-beta-carboxymuconic-epsilon-semialdehyde decarboxylase (ACMSD) is critically dependent on a transition metal ion [Li, T., Walker, A. L., Iwaki, H., Hasegawa, Y., and Liu, A. (2005) J. Am. Chem. Soc. 127, 12282-12290]. Sequence analysis in this study further suggests that ACMSD belongs to the amidohydrolase superfamily, whose structurally characterized members comprise a catalytically essential metal cofactor. To identify ACMSD's metal ligands and assess their functions in catalysis, a site-directed mutagenesis analysis was conducted. Alteration of His-9, His-177, and Asp-294 resulted in a dramatic loss of enzyme activity, substantial reduction of the metal-binding ability, and an altered metallocenter electronic structure. Thus, these residues are confirmed to be the endogenous metal ligands. His-11 is implicated in metal binding because of the strictly conserved HxH motif with His-9. Mutations at the 228 site yielded nearly inactive enzyme variants H228A and H228E. The two His-228 mutant proteins, however, exhibited full metal-binding ability and a metal center similar to that of the wild-type enzyme as shown by EPR spectroscopy. Kinetic analysis on the mutants indicates that His-228 is a critical catalytic residue along with the metal cofactor. Since the identified metal ligands and His-228 are present in all known ACMSD sequences, it is likely that ACMSD proteins from other organisms contain the same cofactor and share similar catalytic mechanisms. ACMSD is therefore the first characterized member in the amidohydrolase superfamily that represents a C-C breaking activity.     

A plant growth-promoting pseudomonad is closely related to the Pseudomonas syringae complex of plant pathogens

Pseudomonas putida GR12-2 is well known as a plant growth-promoting rhizobacterium; however, phylogenetic analysis using the 16S rRNA gene and four housekeeping genes indicated that this strain forms a monophyletic group with the Pseudomonas syringae complex, which is composed of several species of plant pathogens. On the basis of these sequence analyses, we suggest that P. putida GR12-2 be redesignated as P. syringae GR12-2. To compare the ecological roles of P. syringae GR12-2 with its close relatives P. syringae pathovar (pv.) tomato DC3000 and P. syringae pv. syringae B728a, we investigated their ability to cause disease and promote plant growth. When introduced on tobacco or tomato leaves, P. syringae GR12-2 was unable to elicit a hypersensitive response or cause disease, which are characteristic responses of P. syringae DC3000 and B728a, nor were type III secretion system genes required for virulence detected in P. syringae GR12-2 by PCR or DNA hybridization. In contrast to P. syringae GR12-2, neither of the phytopathogens was able to promote root growth when inoculated onto canola seeds. Although commensals and nonpathogens have been reported among the strains of the P. syringae complex, P. syringae GR12-2 is a mutualist and a phytostimulator.     

Primary structure of a ribonuclease from porcine liver, a new member of the ribonuclease superfamily

In most tissues, ribonucleases (RNases) are found in a latent form complexed with ribonuclease inhibitor (RI). To examine whether these so-called cytoplasmic RNases belong to the same superfamily as pancreatic RNases, we have purified from porcine liver two such RNases (PL1 and PL3) and examined their primary structures. It was found that RNase PL1 belonged to the same family as human RNase Us [Beintema et al. (1988) Biochemistry 27, 4530-4538] and bovine RNase K2 [Irie et al. (1988) J. Biochem. (Tokyo) 104, 289-296]. RNase PL3 was found to be a hitherto structurally uncharacterized type of RNase. Its polypeptide chain of 119 amino acid residues was N-terminally blocked with pyroglutamic acid, and its sequence differed at 63 positions with that of the pancreatic enzyme. All residues important for catalysis and substrate binding have been conserved. Comparison of the primary structure of RNase PL3 with that of its bovine counterpart (RNase BL4; M. Irie, personal communication) revealed an unusual conservation for this class of enzymes; the 2 enzymes were identical at 112 positions. Moreover, comparison of the amino acid compositions of these RNases with that of a human colon carcinoma-derived RNase, RNase HT-29 [Shapiro et al. (1986) Biochemistry 25, 7255-7264], suggested that these three proteins are orthologous gene products. The structural characteristics of RNases PL1 and PL3 were typical of secreted RNases, and this observation questions the proposed cytoplasmic origin of these RI-associated enzymes.     

SIRE-1, A putative plant retrovirus is closely related to a legume TY1-copia retrotransposon family

SIRE-1 is a potential soybean retrovirus which has a gene order similar to Ty1-copia retrotransposons but also contains an envelope-like open reading frame (ORF), which is characteristic of retroviruses. PCR and Southern analysis reveals that SIRE-1 is closely related to a legume-wide family of envelope-lacking Ty1-copia group retrotransposons which suggests that SIRE-1 was formed by the recent acquisition of an envelope gene by a Ty1-copia retrotransposon.     

Molecular cloning of gp49, a cell-surface antigen that is preferentially expressed by mouse mast cell progenitors and is a new member of the immunoglobulin superfamily.

gp49 is a Mr 49,000 glycoprotein expressed on the surface of mouse bone marrow-derived mast cells, which are progenitors for the major in vivo mast cell subclasses, typified by intestinal mucosal mast cells and serosal mast cells. The amino-terminal amino acid sequence of gp49 was determined after isolation of the solubilized membrane protein by affinity chromatography with the B23.1 anti-gp49 monoclonal antibody. Redundant oligonucleotides were used to isolate a full-length 1.3-kilobase cDNA from a mouse mast cell library. The predicted amino acid sequence contains a signal peptide of 23 residues, an extracellular domain of 215 residues with three potential sites of N-linked glycosylation, a transmembrane domain of 23 residues, and a cytoplasmic tail of 42 residues. Hybridization of the gp49 cDNA was limited to mRNA extracted from those cell types that also bound the B23.1 monoclonal antibody as assessed by cytofluorographic analyses. The predicted extracellular domain of gp49 contains two regions of 48 and 51 amino acids, each flanked by cysteine residues. Both regions meet criteria for being C2-type domains of the immunoglobulin superfamily based upon the alignment of consensus amino acids and their predicted secondary structure organization. Thus, gp49, a membrane glycoprotein preferentially expressed by the progenitor mast cell population, is a new member of the immunoglobulin superfamily.     

Theiler''s virus genome is closely related to that of encephalomyocarditis virus, the prototype cardiovirus.

Theiler's virus causes a persistent demyelinating infection of the mouse central nervous system. Our study of the molecular mechanism of persistence led us to sequence 1925 nucleotides located at the 3' end of the viral genome. We observed extensive homologies between this region and the corresponding region of encephalomyocarditis virus, the prototype cardiovirus, and only some homologies with the 3' ends of foot-and-mouth disease virus, rhinovirus, and poliovirus genomes.     

Structural evidence that brain cyclic nucleotide phosphodiesterase is a member of the 2H phosphodiesterase superfamily

2',3'-Cyclic-nucleotide 3'-phosphodiesterase (CNP) is an enzyme abundantly present in the central nervous system of mammals and some vertebrates. In vitro, CNP specifically catalyzes the hydrolysis of 2',3'-cyclic nucleotides to produce 2'-nucleotides, but the physiologically relevant in vivo substrate remains obscure. Here, we report the medium resolution NMR structure of the catalytic domain of rat CNP with phosphate bound and describe its binding to CNP inhibitors. The structure has a bilobal arrangement of two modules, each consisting of a four-stranded beta-sheet and two alpha-helices. The beta-sheets form a large cavity containing a number of positively charged and aromatic residues. The structure is similar to those of the cyclic phosphodiesterase from Arabidopsis thaliana and the 2'-5' RNA ligase from Thermus thermophilus, placing CNP in the superfamily of 2H phosphodiesterases that contain two tetrapeptide HX(T/S)X motifs. NMR titrations of the CNP catalytic domain with inhibitors and kinetic studies of site-directed mutants reveal a protein conformational change that occurs upon binding.     

A novel plant ferritin subunit from soybean that is related to a mechanism in iron release

Ferritin is a multimeric iron storage protein composed of 24 subunits. Ferritin purified from dried soybean seed resolves into two peptides of 26.5 and 28 kDa. To date, the 26.5-kDa subunit has been supposed to be generated from the 28-kDa subunit by cleavage of the N-terminal region. We performed amino acid sequence analysis of the 28-kDa subunit and found that it had a different sequence from the 26.5-kDa subunit, thus rendering it novel among known soybean ferritins. We cloned a cDNA encoding this novel subunit from 10-day-old seedlings, each of which contained developed bifoliates, an epicotyl and a terminal bud. The 26.5-kDa subunit was found to be identical to that identified previously lacking the C-terminal 16 residues that correspond to the E helix of mammalian ferritin. However, the corresponding region in the 28-kDa soybean ferritin subunit identified in this study was not susceptible to cleavage. We present evidence that the two different ferritin subunits in soybean dry seeds show differential sensitivity to protease digestions and that the novel, uncleaved 28-kDa ferritin subunit appears to stabilize the ferritin shell by co-existing with the cleaved 26.5-kDa subunit. These data demonstrate that soybean ferritin is composed of at least two different subunits, which have cooperative functional roles in soybean seeds.