Studies on hemagglutinins (lectins) from plasma of murrel fish, family Channidae

Hemagglutinating activity can be identified in the plasma of different species of murrel fish. This activity may be divided into four types according to their agglutinability towards erythrocytes from different sources. Type I plasma agglutinates human blood group A erythrocytes, type II can agglutinate neuraminidase treated human A B O erythrocytes, type III shows no agglutinating activity towards human erythrocytes, while type IV agglutinates human erythrocytes non-specifically. All of them bind to DEAE-cellulose but elute out by different salt concentrations. Type IV plasma is found to be a combination of three separate hemagglutinins, which are separable by sequential binding to human A B O erythrocytes. Blood group A specific lectin activity is purified from this plasma using formalinised A group erythrocytes. The apparent homogeneity of this purified lectin is established by polyacrylamide gel electrophoresis, isoelectric focusing and immunodiffusion. This agglutinin is antigenically identical with that isolated from type I plasma by affinity chromatography on N-acetyl-D-galactosamine coupled to epoxy-activated cellulose column. Their molecular weights are also found to be identical (Mr 140,000) in polyacrylamide gel electrophoresis, having two identical subunits. Forssman glycolipid (0.03 mM) was found to be the most potent inhibitor of agglutination, although Gal beta 1-3 GalNAc (0.09 mM) is also a good inhibitor. Exhaustive dialysis of the purified lectin (hemagglutinin) against EDTA denatures it irreversibly by dissociating it to its subunit structure. Thus human A group agglutinating activity isolated from type I and type IV plasma are identical.     

Metalloproteinase inhibitors from bovine cartilage and body fluids

Inhibitors of the mammalian metalloproteinases, collagenase, proteoglycanase and gelatinase were isolated from bovine cartilage (extracts and culture medium) and bovine amniotic fluid and serum. These inhibitors either bind or do not bind to concanavalin-A--Sepharose, with Mr (gel filtration) of about 30 000 and 20 000, respectively. Cartilage and chondrocyte culture media contained only concanavalin-A-binding inhibitors whereas cartilage extracts contained only a non-binding inhibitor: serum and amniotic fluid contained both forms of inhibitory activities. In moist biochemical respects, particularly in their abilities to inhibit metalloproteinases, all of the inhibitors were found to be similar. It is concluded that the forms of the inhibitors that differ in Mr may be closely related to the tissue inhibitor of metalloproteinases (TIMP) previously purified from rabbit and human sources. These findings help to clarify other studies on collagenase inhibitors and support the concept that TIMP-like inhibitors may be important in the control of connective tissue degradation.     

Inhibition of type I collagen film degradation by tumour cells using a specific antibody to collagenase and the specific tissue inhibitor of metalloproteinases (TIMP)

Rabbit VX2 tumour cells in culture produced a collagenolytic activity which was shown to be immunologically identical to collagenase from rabbit articular chondrocytes and bone. VX2 cells degraded type I collagen films spontaneously and did not produce detectable levels of the tissue inhibitor of metalloproteinases (TIMP). Chondrocytes, however, required both stimulation of collagenase synthesis and activation to effect lysis and were observed to make appreciable amounts of TIMP. The degradation of type I collagen films by VX2 tumour cells was significantly inhibited by both a specific antibody to rabbit collagenase and by purified TIMP, thus demonstrating the unequivocal role of collagenase in this model system.     

Purification and sequence analysis of two rat tissue inhibitors of metalloproteinases.

Two protein inhibitors of metalloproteinases (TIMP) were isolated from medium conditioned by the clonal rat osteosarcoma line UMR 106-01. Initial purification of both a 30-kDa inhibitor and a 20-kDa inhibitor was accomplished using heparin-Sepharose chromatography with dextran sulfate elution followed by DEAE-Sepharose and CM-Sepharose chromatography. Purification of the 20-kDa inhibitor to homogeneity was completed with reverse-phase high-performance liquid chromatography. The 20-kDa inhibitor was identified as rat TIMP-2. The 30-kDa inhibitor, although not purified to homogeneity, was identified as rat TIMP-1. Amino terminal amino acid sequence analysis of the 30-kDa inhibitor demonstrated 86% identity to human TIMP-1 for the first 22 amino acids while the sequence of the 20-kDa inhibitor was identical to that of human TIMP-2 for the first 22 residues. Treatment with peptide:N-glycosidase F indicated that the 30-kDa rat inhibitor is glycosylated while the 20-kDa inhibitor is apparently unglycosylated. Inhibition of both rat and human interstitial collagenase by rat TIMP-2 was stoichiometric, with a 1:1 molar ratio required for complete inhibition. Exposure of UMR 106-01 cells to 10(-7) M parathyroid hormone resulted in approximately a 40% increase in total inhibitor production over basal levels.     

Purified human chondroitin-4-sulfate reduced MMP/TIMP imbalance induced by iron plus ascorbate in human fibroblast cultures

Imbalance between matrix metalloproteinases (MMPs) and tissue inhibitor of matrix metalloproteinases (TIMPs) is an important control point in tissue remodelling. Several findings have reported a marked MMP/TIMP imbalance in a variety of in vitro models in which oxidative stress was induced. Since previous studies showed that commercial hyaluronan and chondroitin-4-sulphate are able to limit lipid peroxidation during oxidative stress, we investigated the antioxidant capacity of purified human plasma chondroitin-4-sulfate in reducing MMP and TIMP imbalance in a model of ROS-induced oxidative injury in fibroblast cultures. Purified human plasma chondroitin-4-sulfate was added to the fibroblast cultures exposed to FeSO4 plus ascorbate. We assayed cell death, MMP and TIMP mRNA expression and protein activities, DNA damage, membrane lipid peroxidation, and aconitase depletion. FeSO4 plus ascorbate produced severe death of cells and increased MMP-1, MMP-2 and MMP-9 expression and protein activities. It also caused DNA strand breaks, enhanced lipid peroxidation and decreased aconitase. TIMP-1 and TIMP-2 protein levels and mRNA expression remain unaltered. Purified human plasma C4S, at three different doses, restored the MMP/TIMP homeostasis, increased cell survival, reduced DNA damage, inhibited lipid peroxidation and limited impairment of aconitase. These results further support the hypothesis that these biomolecules possess antioxidant activity and by reducing ROS production C4S may limit cell injury produced by MMP/TIMP imbalance.     

Tissue inhibitor of metalloproteinase-2 (TIMP-2) has erythroid-potentiating activity.

Tissue inhibitor of metalloproteinase (TIMP) was purified and molecularly cloned on the basis of its erythroid-potentiating activity (EPA). TIMP/EPA appears to be a bifunctional molecule with both growth factor and anti-enzymatic activity. Recently, a second TIMP-related molecule was identified and we have investigated its possible erythroid-potentiating activity. Native, purified human TIMP-2 was assayed for erythroid-potentiating activity using an in vitro erythroid burst formation assay and was compared with that of previously characterized recombinant EPA/TIMP-1. The results demonstrate that both members of the tissue inhibitor of metalloproteinase family, TIMP-1 and TIMP-2, possessed erythroid potentiating activity which was inhibited by antibodies developed to neutralize EPA. These results suggest that TIMP-2 shares a common structural domain with EPA/TIMP-1 that is responsible for the erythroid-potentiating activity of these inhibitors. Therefore, TIMP-1 and TIMP-2, with both anti-protease activity and growth factor activity, join a family of bifunctional molecules such as fibroblast growth factor and thrombin which have both enzymatic and growth factor activity.     

人TIMP—3cDNA的克隆,表达及其抗血管生成作用

从人新鲜的胎盘组织中提取总ＲＮＡ,以ＲＴ－ＰＣＲ法获取了人组织金属蛋白酶抑制－３成熟蛋白的ｃＤＮＡ。序列分析表明,ＴＩＭＰ－３成熟蛋白含有１８８个氨基酸残基,其中１２个半胱氨酸残基在ＴＩＭＰ家族中高度保守。将人ＴＩＭＰ－３ｃＤＮＡ插入含７启动子的质粒ｐＥＴ－２４构建表达质粒ｐＥＴ－ＴＩＭＰ３,转化大肠杆菌ＢＬ２１,筛选表达菌株ＢＬＴＩＭＰ３。     

Isolation of bovine angiogenin using a placental ribonuclease inhibitor binding assay

Angiogenin, which induces the formation of new blood vessels, was isolated previously from two human sources--HT-29 tumor conditioned media and normal plasma. By use of a newly developed binding assay, a similar protein has now been purified from bovine plasma at levels of 30-80 micrograms/L. This protein has the structural, enzymatic, and biological characteristics expected for an angiogenin molecule. Its amino acid composition is similar to that of the human protein, and 22 of 31 residues in the amino-terminal sequences are identical, including a block of 11 consecutive residues. Like human angiogenin, the bovine protein binds placental ribonuclease inhibitor, is inactive toward conventional RNase A substrates, and displays selective ribonucleolytic activity toward some rRNAs. In addition, the bovine protein induces angiogenesis in vivo in the chick embryo chorioallantoic membrane assay at levels as low as 44 fmol per egg. Thus, angiogenin is present in bovine sera at levels similar to those observed in man, and its enzymatic and biological activities are identical with those of the human protein.     

Engineering of TIMP‐3 as a LAP‐fusion protein for targeting to sites of inflammation

Tissue inhibitor of metalloproteinase (TIMP)‐3 is a natural inhibitor of a range of enzymes that degrade connective tissue and are involved in the pathogenesis of conditions such as arthritis and cancer. We describe here the engineering of TIMP‐3 using a novel drug‐delivery system known as the ‘LAP technology’. This involves creating therapeutic proteins in fusion with the latency‐associated peptide (LAP) from the cytokine TGF‐? to generate proteins that are biologically inactive until cleavage of the LAP to release the therapy. LAP‐TIMP‐3 was successfully expressed in mammalian cells and the presence of the LAP resulted in a 14‐fold increase in the quantity of recombinant TIMP‐3 produced. LAP‐TIMP‐3 was latent until release from the LAP by treatment with matrix metalloproteinase when it could inhibit proteases of the adamalysins and adamalysins with thrombospondin motifs families, but not matrix metalloproteinases, indicating that this version of TIMP‐3 is a more specific inhibitor than the native protein. There was sufficient protease activity in synovial fluid from human joints with osteoarthritis to release TIMP‐3 from the LAP fusion. These results demonstrate the potential for development of TIMP‐3 as a novel therapy for conditions where upregulation of catabolic enzymes are part of the pathology.     

Cloning and developmental regulation of tissue inhibitor of metalloproteinases-3 (TIMP3) in Xenopus laevis early embryos

We cloned a cDNA encoding tissue inhibitor of metalloproteinases-3 (TIMP3) from the frog Xenopus laevis. Similar to TIMP3 from other species, Xenopus TIMP3 has 188 residues including 12 conserved cysteines and Asn¹⁸⁴, a putative site for N-linked sugars. Xenopus TIMP3 is 84% identical with human TIMP3. As shown by Northern blotting and RT-PCR, Xenopus TIMP3 mRNA is maternally inherited in eggs and midblastula (stage 8) embryos, downregulated in gastrula and then upregulated in neurula and pretailbud embryos. In select adult tissues, TIMP3 mRNA is present in heart, muscle, liver, skin, intestine and ovaries. These results suggest that TIMP3 is involved in the regulation of expression of matrix metalloproteinases in Xenopus early development and adult tissue remodeling.     

Purification and characterization of bovine interstitial collagenase and tissue inhibitor of metalloproteinases.

In this report we describe the purification of bovine interstitial collagenase and provide information on its substrate specificity, kinetic parameters of catalytic activity, and amino terminal protein sequence. In addition, we present a simplified protocol for the purification of bovine tissue inhibitor of metalloproteinases (TIMP). Collagenase was purified by sequential chromatography through heparin-Sepharose, DEAE-Sepharose, and green-agarose, resulting in a product that was greater than 95% pure as judged by polyacrylamide electrophoresis. Typical of other interstitial collagenases, the isolated bovine protein was activated by protease and organomercurial treatment. It also demonstrated a kinetics and substrate specificity similar to those of human collagenase. TIMP was purified by sequential chromatography through heparin-Sepharose and DEAE-Sepharose followed by reverse-phase HPLC. The purified protein had a size, N-terminal sequence, and inhibitor activity similar to those of other mammalian TIMPs. Partial peptide sequences suggested that bovine collagenase and TIMP have strong sequence homology to their human homologues.     

Identification of plasma kallikrein as an activator of latent collagenase in rheumatoid synovial fluid

Rheumatoid synovial fluid contains an activator of latent collagenase from culture medium of pig synovium. The activator was purified by gel chromatography on Ultrogel AcA 44 and affinity chromatography on soybean trypsin inhibitor coupled to Sepharose 4B. The purified material was homogeneous on SDS-polyacrylamide gel electrophoresis with Mr 88 000. The activator had limited proteolytic activity against azo-casein, but showed amidase activity on Pro-Phe-Arg-NMec, Z-Phe-Arg-NMec, D-Val-Leu-Arg-NPhNO2 and D-Pro-Phe-Arg-NPhNO2, with an optimum at pH 8.0. Activity was completely inhibited by diisopropyl fluorophosphate, soybean trypsin inhibitor, leupeptin and Pro-Phe-Arg-CH2Cl, whereas lima bean trypsin inhibitor, Tos-Lys-CH2Cl, a specific inhibitor of factor XIIa from maize, EDTA and iodoacetate were not inhibitory. These properties of the activator suggested that it might be plasma kallikrein (EC 3.4.21.34), and the possibility was further examined. The activator was treated with [3H]diisopropyl fluorophosphate, and run in SDS-polyacrylamide gel electrophoresis with reduction; a radioautograph of the gel showed a pair of [3H]diisopropyl phosphoryl-labelled bands (Mr 36 000 and 34 000) identical to those obtained with authentic plasma kallikrein. Double immunodiffusion with monospecific antiserum against human plasma kallikrein confirmed the identification. This is the first demonstration of collagenase-activating activity of plasma kallikrein, and raises the possibility that activation of prokallikrein in the inflamed joint space may contribute to the disease process not only by the production of bradykinin, but also by activating latent collagenase.     

Isolation and characterization of tissue inhibitors of metalloproteinases (TIMP-1 and TIMP-2) from human rheumatoid synovial fluid.

The tissue inhibitors of metalloproteinases TIMP-1 and TIMP-2 were purified to apparent homogeneity from human rheumatoid synovial fluid (HRSF). The inhibitors were isolated by dissociation of non-covalent gelatinase/TIMP complexes. TIMP-1 migrated as a single polypeptide with Mr 28,500 on SDS-PAGE, while the Mr of TIMP-2 was 21,000. The inhibitory activity was stable under heat and acid pH. N-terminal sequence data were obtained for the first 15 residues of both inhibitors and showed identity to the human fibroblast inhibitors TIMP-1 and TIMP-2. This is the first demonstration that TIMP-1 and TIMP-2 can be directly purified from human rheumatoid synovial fluid. The complex formation between the metalloproteinase inhibitors and leucocyte metalloproteinases was shown by immunoblotting.     

The degradation of bovine parathyroid hormone by leukocytes

Human peripheral leukocytes catalyse an elastase-like cleavage of bovine parathyroid hormone, with an identical specificity to that previously observed with a neutral proteinase (EC 3.4.21-) isolated from the outer surface of plasma membranes from human leukocytes. Parathyroid hormone is not hydrolysed by human erythrocytes, and polymorphonuclear leukocytes are much more effective in hormone degradation than lymphocytes. Despite the fact that purified human leukocyte granular elastase (EC 3.4.21.37) catalyses an identical cleavage reaction, the hydrolysis observed with intact cells is clearly not due to proteinases secreted by the cells. The reaction is inhibited by human serum and by purified human alpha-1-antitrypsin, but not by antibodies to human leukocyte granular elastase. The possible significance of these phenomena to the in vivo metabolism of parathyroid hormone are discussed.     

Polymorphic X-chromosome inactivation of the human TIMP1 gene.

X inactivation silences most but not all of the genes on one of the two X chromosomes in mammalian females. The human X chromosome preserves its activation status when isolated in rodent/human somatic-cell hybrids, and hybrids retaining either the active or inactive X chromosome have been used to assess the inactivation status of many X-linked genes. Surprisingly, the X-linked gene for human tissue inhibitor of metalloproteinases (TIMP1) is expressed in some but not all inactive X-containing somatic-cell hybrids, suggesting that this gene is either prone to reactivation or variable in its inactivation. Since many genes that escape X inactivation are clustered, we examined the expression of four genes (ARAF1, ELK1, ZNF41, and ZNF157) within approximately 100 kb of TIMP1. All four genes were expressed only from the active X chromosome, demonstrating that the factors allowing TIMP1 expression from the inactive X chromosome are specific to the TIMP1 gene. To determine if this variable inactivation of TIMP1 is a function of the hybrid-cell environment or also is observed in human cells, we developed an allele-specific assay to assess TIMP1 expression in human females. Expression of two alleles was detected in some female cells with previously demonstrated extreme skewing of X inactivation, indicating TIMP1 expression from the inactive chromosome. However, in other cells, no expression of TIMP1 was observed from the inactive X chromosome, suggesting that TIMP1 inactivation is polymorphic in human females.     

Specific inhibitor of complement (C5)-derived chemotactic activity in systemic lupus erythematosus related antigenically to the Bb fragment of human factor B

Serum and plasma from patients with active systemic lupus erythematosus contain a specific inhibitor of complement (C5)-derived chemotactic activity. We found that the inhibitor is antigenically related to the Bb fragment of complement factor B. Lupus plasma and purified inhibitor significantly reduced the chemotactic activity of zymosan-treated normal serum, an effect that was abolished by antibodies to factor B. Similar results were obtained when purified Bb was used. Neither purified inhibitor nor Bb inhibited the chemotactic activity of purified human C5a or C5a des Arg. As reported previously, the chemotactic activity of C5a des Arg was enhanced significantly by the addition of an anionic polypeptide (cochemotaxin) present in normal serum and plasma. Interestingly, both purified lupus inhibitor and Bb inhibited the chemotactic activity exhibited by mixtures of C5a des Arg and its cochemotaxin. This effect was due, most likely, to their ability to neutralize the enhancing effect of the cochemotaxin on the chemotactic activity of C5a des Arg. Immunoelectrophoresis and western blots revealed that the purified inhibitor reacted with anti-factor B and exhibited a similar charge and molecular weight as purified Bb.     

Proteinase inhibitors of horse seminal plasma. A high molecular mass, acid-soluble proteinase inhibitor

Horse seminal plasma does not possess a proteinase inhibitor corresponding to human HUSI-I (human seminal plasma inhibitor). Instead a protein complex of high relative molecular mass (Mr) containing proteinase inhibitory activity was detected, which was called horse seminal plasma protein complex or HSPC. The compound had a broad enzyme-inhibiting spectrum. Its Mr was estimated to be 800 000 and it was composed of 7 different polypeptides with Mr values ranging from 11 000 to 30 000. Its carbohydrate content was between 3.5% and 5%. Despite the high molecular mass, the complex was soluble in diluted perchloric acid and did not lose its biological activity. The high recovery of seminal plasma protein (69%) after perchloric acid treatment, the unaltered immunoelectrophoretic precipitation pattern of the perchloric acid soluble part of seminal plasma, and the similarity of the polypeptide patterns of unfractionated seminal plasma and HSPC suggest that HSPC is one of the major components of horse seminal plasma. In addition to HSPC, horse seminal plasma contained a group of three electrophoretically distinguishable proteinase inhibitors, corresponding roughly to a Mr of 6500. They inhibited only trypsin. The similar Mr values and the identical narrow enzyme specificity suggest that they are isoinhibitors and may be analogues of human HUSI-II (human seminal plasma inhibitor). The lack of a HUSI-I analog in the horse is discussed in relation to a previously made observation that horse tracheobronchial fluid contains no detectable perchloric acid-soluble proteinase inhibitors.     

The 21-kDa protein is a transformation-sensitive metalloproteinase inhibitor of chicken fibroblasts.

We report the electrophoretic purification and characterization of the 21-kDa protein, an extracellular matrix component synthesized during the early stages of transformation of chicken embryo fibroblasts infected with Rous sarcoma virus (Blenis, J., and Hawkes, S. P. (1983) Proc. Natl. Acad. Sci. U. S. A. 80, 770-774; Blenis, J., and Hawkes, S. P. (1984) J. Biol. Chem. 259, 11563-11570). The NH2-terminal amino acid sequence of the protein is greater than 60% identical to a consensus sequence of mammalian tissue inhibitor of metalloproteinases (TIMP). It shares several biochemical properties with other metalloproteinase inhibitors, including evidence of intrachain disulfide bonds and resistance to cleavage by trypsin. An electrophoretic assay employing a metal ion-dependent gelatinase from conditioned cell culture medium demonstrates inhibitor activity for purified 21-kDa protein. The 21-kDa protein is the major inhibitor in the extracellular matrix and appears unique in solubility properties among inhibitors with a TIMP-like sequence. Statistical analysis of amino acid composition data for these inhibitors defines two distinct groups (TIMP and TIMP-2) and supports a close relationship for the 21-kDa protein with the TIMP group. However, the apparent size and lack of glycosylation align it more closely with the TIMP-2 group of proteins. Therefore, it is possible that the 21-kDa protein is a variant of TIMP or, alternatively, represents a third protein within the metalloproteinase inhibitor family. This report provides the first evidence that avian metalloproteinase inhibitors are similar in sequence to their mammalian counterparts.     

Bovine plasma protein C inhibitor with structural and functional homologous properties to human plasma protein C inhibitor

Bovine plasma protein C inhibitor was purified; it was then characterized in comparison with human protein C inhibitor. The specific inhibitory activity of the purified inhibitor for bovine activated protein C was 8,500 times that of the inhibitor in plasma. The purified inhibitor showed a single band with Mr 56,000 by SDS-PAGE at pH 7.0, and two bands at pH 8.8, a major one with Mr 56,000 and a minor one with Mr 105,000, under both unreduced and reduced conditions. The pI range of the inhibitor was between 4.4 and 6.1. The Mr of the inhibitor was reduced by treatment with neuraminidase, O-glycanase, and also with glycopeptidase-A, suggesting that the inhibitor has both Asn-linked and Ser/Thr-linked carbohydrate chains. Twenty-seven of the NH2-terminal 49 amino acid residues of the bovine inhibitor, which lacks the first 4 residues from the NH2-terminal amino acid sequence of human inhibitor, were identical to those of the human inhibitor. The bovine inhibitor inhibited bovine and human activated protein C, human thrombin, Factor Xa, Factor XIa, and plasma kallikrein with Ki = 1.0, 5.2, 2.6, 3.0, 1.3 X 10(-8) M, and 4.5 X 10(-9) M, respectively. The inhibitory rates for activated protein C and thrombin were accelerated significantly in the presence of heparin or negatively charged dextran sulfate. However, the acceleration by heparin or dextran sulfate for the inhibition of Factor Xa, Factor XIa, and plasma kallikrein was not significant. The bovine inhibitor did not inhibit human Factor XIIa or plasmin.(ABSTRACT TRUNCATED AT 250 WORDS)