A fibronectin-binding protein from rice bran with cell adhesion activity for animal tumor cells.

A rice bran 57-kDa protein was isolated by affinity chromatography with fibronectin immobilized on agarose. This fibronectin-binding protein designated as RB-57 had an amino-terminal amino acid sequence identical with that of a putative mature form of rice hydroxyproline-rich glycoprotein. A distinct feature of the amino acid composition of RB-57 was the high contents of hydroxyproline and proline representing about 45% of the total amino acids. The sugar analysis indicated that arabinose represented 46.8% of the total carbohydrates. RB-57 showed cell adhesion activity for murine Lewis lung carcinoma cells. The result suggests that RB-57 may play a role in plant cell adhesion, although cell adhesion-promoting activity for plant cells remains to be tested.

Isolation of a 41-kDa protein with cell adhesion activity for animal tumor cells from the mushroom Hypsizigus marmoreus by affinity chromatography with type IV collagen immobilized on agarose.

A type IV collagen-binding protein of 41 kDa was isolated from the mushroom Hypsizigus marmoreus and the protein was designated as HM41. The Western blotting analysis with anti-HM41 antibodies demonstrated that HM41 was unrelated to HM23, which had been shown to have an affinity for type IV collagen. The microsequence analysis of the membrane-blotted peptides generated by fragmentation with cyanogen bromide showed no homologous proteins reported. HM41 had cell adhesion-promoting activity for murine Lewis lung carcinoma LL2 cells and human fibrosarcoma HT1080 cells. These results indicate that HM41 is a hitherto undescribed fungus protein that can interact both with animal extracellular matrix protein type IV collagen and with animal tumor cells.

Nucleotide sequence of gene PBII encoding salivary proline-rich protein P-B.

The nucleotide sequence of gene PBII encoding salivary proline-rich protein P-B was determined. PBII is 7.1 kb long and contains 3 exons. PBII exhibits considerable nucleotide sequence homology not only in exons but also in introns with PBI (accession number D89501), the gene whose nucleotide sequence was determined previously [Isemura and Saitoh (1997) J. Biochem. 121, 1025-1030]. PBI and II constitute a gene family distinct from that to which the majority of salivary proline-rich protein ones belong. The nucleotide sequence data reported in this paper will appear in the DDBJ, EMBL, and GenBank nucleotide sequence databases under accession number AB031740.

Cell adhesion activity for murine carcinoma cells of a wheat germ 55-kDa protein with binding affinity for animal extracellular matrix proteins.

A wheat germ 55-kDa protein was isolated by affinity chromatography with Matrigel immobilized on agarose, followed by preparative gel electrophoresis. This Matrigel-binding protein designated as WG-55 had an amino-terminal amino acid sequence which is identical to that of a putative mature form of wheat storage protein Gbl 1. WG-55 reacted with concanavalin A, indicating its glycoprotein nature as expected from the amino acid sequence of Gbl 1. As expected, similarly, WG-55 exhibited RGD-dependent cell adhesion activity for murine carcinoma cells. These data suggest that WG-55 or mature Gbl 1 protein may play a role in plant cell adhesion.

Interaction between the carboxyl-terminal heparin-binding domain of fibronectin and (-)-epigallocatechin gallate.

We have previously reported that (-)-epigallocatechin gallate (EGCg) inhibited lung carcinoma cell adhesion to fibronectin (FN) and demonstrated its interaction with FN. In the present work, we studied the interaction between thermolysin fragments of FN and EGCg. An amino acid sequence analysis of the fragment bound by EGCg-agarose provided its identification as a carboxyl-terminal heparin-binding domain. Thus, the inhibition of cancer cell adhesion to FN by EGCg is not caused by its direct binding to the cell-binding domain containing an Arg-Gly-Asp-sequence.

Bilirubin binding activity of cytokeratin 18 isolated from the porcine liver.

A porcine liver 40 kDa protein designated SBP40 isolated by affinity chromatography with agarose-linked spermine was identified as a porcine cytokeratin 18 on the basis of partial amino acid sequences of peptides derived by lysylendopeptidase digestion and by its reactivity with two commercially available preparations of monoclonal antibody. Immunohistochemistry revealed that SBP40 is localized at the hepatocyte membranes, preferentially in the bile canalicular area in accordance with the previously reported localization of cytokertain 18 in the murine liver. Affinity chromatography with agarose-linked bilirubin, a solubilization experiment of bilirubin from bilirubin-Sephadex G-10 complex, and gel-filtration of a mixture with bilirubin demonstrated that SBP40 or porcine cytokeratin 18 has binding affinity for bilirubin. These results suggest that cytokeratin 18 may play a role as a membrane reservoir in the event of transport and secretion of bile pigments in the liver.

Nucleotide sequence of gene PBI encoding a protein homologous to salivary proline-rich protein P-B.

The nucleotide sequence of gene PBI, the putative translational product of which is homologous to salivary proline-rich protein P-B, has been determined. PBI is 6.4 kb long and contains 3 exons. PBI appears to code for the precursor of a proline-rich protein which we have designated P-B1. The P-B1 precursor is composed of 134 amino acid residues including 22 residues of signal sequence, 23 residues of N-terminal sequence, five repeating units of 13 to 14 residues and 22 residues of C-terminal sequence. The signal sequence of this precursor is identical with that of the P-B precursor. The N-terminal 61 residue sequence of P-B1 has homology of 75% with the whole sequence of P-B (57 residues), when deletion of 4 residues is taken into account. P-B1 is 55 residues longer than P-B.

Production of human salivary type cysteine proteinase inhibitors (cystatins) by an Escherichia coli system and partial characterization of recombinant cystatin S and its mutant (117 arginine-->tryptophan).

The cDNAs encoding the precursors of cystatin SN, cystatin S, and two mutants of cystatin S (-18R-->W; 117R-->W) were expressed in Escherichia coli JM109 with isopropyl-beta-D-thio-galactoside (IPTG) induction. Premature cystatin S with the original signal [-20MARPLCTLLLLMATLAGALA] was processed and a large amount of the mature form was produced. A mutation (-18R-->W) in the signal reduced its accumulation in periplasmic space remarkably. The amount of cystatin SN accumulated in the periplasm was slightly smaller than that of cystatin S. The periplasmic fraction was prepared by cold osmotic-shock treatment and the expressed cystatins were detected using anti-cystatin S antibody. Recombinant cystatin S and its mutant (117R-->W) were purified from the periplasmic fractions with an ion exchange column of DEAE-cellulose. The amino (N-) terminal 10 residues of recombinant cystatin S was sequenced to be SSSKEENRII-, which is exactly identical to that of the authentic mature cystatin S. Recombinant cystatin S and the mutant showed virtually the same inhibitory properties for ficin, papain and cathepsin B as the native cystatin S and its monophosphorylated form. The inhibitory activity of the both recombinant cystatins for cathepsin C was weaker than those of the native cystatin S and phosphorylated cystatin S.

Genetic polymorphisms of the CST2 locus coding for cystatin SA.

A new genetic polymorphism of cystatin SA has been identified in human submandibular-sublingual saliva by means of basic gel electrophoresis and immunoblotting with anti-cystatin S. Two proteins, SA1 and SA2, are given by two alleles of CST2, viz., CST2*1 and CST*2. Inheritance is controlled by two codominant alleles at an autosomal locus. This hypothesis is supported by studies of 16 families 32 children. Gene frequencies for CST2*1 and CST2*2 are 0.935 and 0.065, respectively (n = 341). Eighteen amino acids determined among 20 N-terminal residues of cystatin SA2 are identical with the sequence encoded by CST2. Three forms of cystatin S (mono-phosphorylated cystatin S, di-phosphorylated cystatin S, and non-phosphorylated cystatin S) are present in the 341 saliva samples tested.

Inhibitory activities of partially degraded salivary cystatins.

1. Aminoterminally truncated forms of cystatin S and cystatin SN had higher inhibition constants for ficin, but lower ones for cathepsin C (dipeptidyl peptidase I) as compared to their respective full-sized form. 2. Cystatin SN still retained the inhibitory activity for ficin after reduction and carboxymethylation, although the inhibition constant increased.

Molecular cloning and sequence analysis of cDNA coding for the precursor of the human salivary proline-rich peptide P-B.

cDNA coding for the human salivary proline-rich peptide P-B was amplified by polymerase chain reaction using human submaxillary gland cDNA as the template and d(deoxy)T25 as the primer for both strands. The amplified cDNA of 0.8 kbp encodes a hydrophobic signal sequence of 22 amino acid residues and the complete sequence of P-B (57 amino acids). The determined nucleotide sequence of P-B cDNA indicates that P-B is a mature full-size protein and not the enzymic degradation product of a larger protein. The gene coding for P-B is not a member of the proline-rich protein gene family characterized previously.

Molecular biology of human salivary cysteine proteinase inhibitors.

The synthesis of human cystatins of family II is controlled by a multigene family having at least 7 members that are localized on chromosome 20, the cystatin gene family. Proteolytic degradation and phosphorylation of the gene products give rise to heterogeneity and multiplicity of cystatins in human saliva. These cysteine proteinase inhibitors, as well as other members of the cystatin superfamily, are evolutionally and functionally related to Bowman-Birk type serine proteinase inhibitors.

Characterization of two members (CST4 and CST5) of the cystatin gene family and molecular evolution of cystatin genes.

Two members (CST4 and CST5) of the cystatin gene family have been characterized partially by DNA analysis. The CST4 clone contained the gene coding for the precursor form(141 amino acids) of cystatin S, and its exon-intron organization is the same as that of other members (the cystatin SN gene at the CST1 locus, the cystatin SA gene at the CST2 locus, the cystatin C gene at the CST3 locus and a cystatin pseudogene at the CSTP1 locus). The second cystatin pseudogene was elucidated in the clone, CST5, and it was assigned to the CSTP2 locus. Alignment of DNA sequences of cystatin genes with other genes suggested that the genes for cystatins, kininogens, and Bowman-Birk type inhibitors have evolved from an ancient ribonuclease-like gene.

Identification of full-sized forms of salivary (S-type) cystatins (cystatin SN, cystatin SA, cystatin S, and two phosphorylated forms of cystatin S) in human whole saliva and determination of phosphorylation sites of cystatin S.

Our recent work on the gene structures for human salivary (S-type) cystatins [Saitoh, E. et al. (1987) Gene 61, 329-338] has suggested that the structures of cystatins which we determined previously at the protein level lack N-terminal peptide portions of the full-sized intact forms. In the present study, attempts were made to isolate full-sized S-type cystatins by introducing methanol fractionation into the purification steps to suppress the enzymatic activity present in saliva. Full-sized cystatin SN and two phosphorylated forms of full-sized cystatin S were thus isolated. Analysis of one fraction indicated that this was a mixture of full-sized cystatin SA and non-phosphorylated cystatin S. The phosphorylation sites of cystatin S were determined to be Ser-Ser-Ser1(P)-Lys-Glu-Glu- for monophosphorylated cystatin S and Ser1(P)-Ser-Ser3(P)-Lys-Glu-Glu- for diphosphorylated cystatin S. Immunoblotting analysis with anti-cystatin S antiserum revealed that tears and seminal plasma also contained S-type cystatins, but diphosphorylated cystatin S was detected neither in tears nor in seminal plasma and no cystatin SN was found in seminal plasma. These data indicate that S-type cystatins are secreted into the oral cavity without significant degradation in salivary glands or ducts and that they are expressed tissue specifically.

Cystatins of family II are harboring two domains which retain inhibitory activities against the proteinases.

Two cyclic peptides, Ac-CTKSQPNLDTC-NH2 (SA-LOOP1) and Ac-CSFQIYEVPWE DRMSLVNSRC-NH2 (SA-LOOP2) were prepared. These sequences are respectively found in the second and third exons of cystatin SA and are well conserved among the cystatins of family II. In addition, these sequences are extremely homologous to the inhibitory regions of several serine-proteinase inhibitors. The peptides were assayed for their inhibiting properties towards serine- and cysteine-proteinases. SA-LOOP1 inhibited porcine pancreatic trypsin (Ki = 370 microM), but did not inhibit cysteine-proteinases. SA-LOOP2 inhibited not only porcine pancreatic alpha-chymotrypsin (Ki = 23 microM) but also papain (Ki = 24 microM) and ficin (Ki = 52 microM). These data indicate that the exon-intron organization of the cystatin genes coinside with the structural and/or functional domains of the protein, and may have significant implications for understanding the active sites of cystatins.

The human cystatin gene family: cloning of three members and evolutionary relationship between cystatins and Bowman-Birk type proteinase inhibitors.

Three genes from the human cystatin gene family have been isolated from a bacteriophage lambda library containing Hind III digests of human genomic DNA. The cloned genes were identified with three DNA probes each containing exon 1, exon 2 and exon 3 of the CST1 gene for cystatin SN. The genes, which we name CST2B, CST4, and CST5, are 6.8 kb, 5.4 kb and 12.5 kb in size, respectively. Statistical analysis of DNA sequence homology elucidated that the second and third exons of cystatin (family II) genes and three cystatin (family II) gene like segments in the kininogen (family III) genes are significantly homologous to the gene segments coding for the inhibitory domains of Bowman-Birk type proteinase inhibitors.

The human cystatin C gene (CST3) is a member of the cystatin gene family which is localized on chromosome 20.

The fourth gene from the human cystatin gene family of salivary-type cysteine-proteinase inhibitors has been isolated and partially characterized by DNA analysis. The gene, which we name CST3, codes for human cystatin C, and has the same organization as the CST1 gene for cystatin SN and the CST2 gene for cystatin SA. Southern analysis of EcoR I digested DNAs from 32 independent somatic cell hybrid clones hybridized to a probe from CST1 demonstrated that all members of the cystatin gene family segregate with human chromosome 20. These results indicate that the genes for salivary-type cystatins and cystatin C are members of a multigene family--the cystatin gene family.

'Salivary peptide P-C' of human pancreatic B-cells shares only partly immunoreactivity with salivary peptide P-C indicating a new B-cell protein which is different from insulin.

Salivary peptide P-C like immunoreactivity, originally isolated from human whole saliva has later been found in the human pancreatic B-cells. In the present work an indirect immunofluorescence technique using monoclonal antibodies against isolated salivary peptide P-C was applied to Bouin fixed pancreas and parotid glands to study the possible identity of the two substances. Positive P-C immunofluorescence was found in the serous cells of parotid glands but not in pancreatic B-cells, suggesting that pancreatic P-C substance is not salivary peptide P-C itself, but a substance sharing the common antigenic site with salivary peptide P-C. To examine this, an indirect immunofluorescence technique using polyclonal P-C antisera pre-absorbed with six kinds of synthetic fragments (1-22, 23-44, 23-29, 30-44, 30-38 and 38-44) of salivary peptide P-C was applied to the human pancreas. The result showed that pancreatic P-C substance was a substance which shares the common antigenic site with the 38-44 amino acid residue of salivary peptide P-C. Western blot analysis using extracts of human pancreata further showed that pancreatic P-C substance is not a precursor of insulin but a protein with molecular weight of 11,500 dalton, indicating the presence of a new protein in the insulin secretory granules of human pancreatic B-cells.