Characterization of matrix metalloproteinase expressed by human embryonic kidney cells

There is little information available on the proteases expressed by human embryonic kidney (HEK) cells, which are often used for expression of recombinant proteins and production of adenovirus vector. The expression profile of proteases in HEK cell line was investigated using zymography, mRNA analysis, western blotting and protein array. The major protease was gelatinase A [or matrix metalloproteinase (MMP)-2]. Beside, other MMPs, such as MMP-1, -2, -3, -8, -9, -10, -13 and membrane type (MT) 1- and 3−MMP, as well as tissue inhibitors of metalloproteinase (TIMP)-1, -2 and -3, were also expressed by HEK cells. Characterization of MMP and TIMP profiles expressed by HEK cells provides the basis for degradation control of recombinant protein and adenovirus vector during culture and purification processes.     

Activation of the matrix metalloproteinase inhibitor complex by a low molecular weight angiogenic factor

Tissue inhibitor of matrix metalloproteinases is the major inhibitor of the collagenolytic enzymes and the inhibitory complex has been thought to be irreversible. In this paper we show that a low molecular weight non-protein endothelial cell stimulating angiogenic factor is able to reactivate the enzyme from the inhibitor complex and liberate free inhibitor. The importance of an angiogenic factor able to initiate limited degradation of extra-cellular matrix such that space is created for new capillary growth is discussed.     

Studies on low molecular weight nuclear protein of tumour and normal cells.

1. Preliminary results of comparative electrophoretical and immunological analyses of the components of two classes of non-histone proteins, i.e. NHP1 and NHP2 eluted from hydroxyapatite allowed to suppose that protein of Mw 18,000 is specific for animal tumour cells. 2. However, the studies on cellular distribution of this polypeptide indicated that it is exclusively located in nuclei of hepatoma and normal liver as well. 3. The former observation seems to be the result of changes of the affinity of this protein to DNA during neoplastic transformation.     

Fibrin-binding urokinase (or precursor form of urokinase) with a molecular weight of about 100,000 in fresh human urine

Fresh human urine was found to contain at least three different molecular forms of fibrin-binding urokinase (UK) or its precursor, all of which were absorbed on a fibrin/Celite column at neutral pH, and could be eluted with 0.3-1.0 mol/l NaCl in phosphate buffer, followed by 0.2 mol/l, Arg, 2 mol/l KSCN, and 2 mol/l urea, respectively. The main molecular form isolated revealed a molecular weight (MW) of approximately 100,000 (UK-100), and the minor ones were estimated to have MW of 150,000-200,000 and 45,000. In contrast, commercially obtained UK preparations contained mostly active enzymes with MW of 53,000 and 32,000, respectively, and the remaining high molecular forms represented less than 2.0% of the total amount. Rabbit monospecific antibody (IgG) against UK subcomponent (active heavy chain; H-chain UK) reacted and inhibited the fibrinolytic activity of all the active UK molecules. The UK-100 isolated was relatively stable in solution at neutral pH and resistant to mild reduction, without molecular change. Although the preparation had a very low specific activity (ca. 300 IU/mg protein), both the pyro-Glu-Gly-Arg-pNA amidolytic and plasminogen activating activities could be partially enhanced by the addition of trace amounts of plasmin. In this process, the appearance of two additional active enzymes of MW 53,000 and 32,000 was also confirmed by zymography.     

Interaction of human rheumatoid synovial collagenase (matrix metalloproteinase 1) and stromelysin (matrix metalloproteinase 3) with human alpha 2-macroglobulin and chicken ovostatin. Binding kinetics and identification of matrix metalloproteinase cleavage sites

The homologous proteinase inhibitors, human alpha 2-macroglobulin (alpha 2M) and chicken ovostatin, have been compared with respect to their "bait" region sequences and interactions with two human matrix metalloproteinases, collagenase and stromelysin. A stretch of 34 amino acid residues of the ovostatin bait region sequence was determined and the matrix metalloproteinase cleavage sites identified. Collagenase cleaved a X-Leu bond where X was unidentified, whereas the major cleavage site by stromelysin was at the Gly-Phe bond, 4 residues on the COOH-terminal side of the collagenase cleavage site. Collagenase cleaved the alpha 2M bait region at the Gly679-Leu680 bond, and stromelysin at Gly679-Leu680 and Phe684-Tyr685 bonds. Sequence similarity in the bait region of members of the alpha-macroglobulin family is strikingly low. The kinetic studies indicate that alpha 2M is a 150-fold better substrate for collagenase than type I collagen. Structural predictions based on the bait region sequences suggest that a collagen-like triple helical structure is not a prerequisite for the efficient binding of tissue collagenase to a substrate. The binding of stromelysin to alpha 2M is slower than that of collagenase. Stromelysin reacts with ovostatin even more slowly. Despite the preference of chicken ovostatin for metalloproteinases, human alpha 2M, a far less selective inhibitor, reacts more rapidly with collagenase and stromelysin. These results suggest that alpha 2M may play an important role in regulating the activities of matrix metalloproteinases in the extracellular space.     

The low density lipoprotein receptor-related protein LRP is regulated by membrane type-1 matrix metalloproteinase (MT1-MMP) proteolysis in malignant cells

We demonstrate that the presentation of LRP and the subsequent uptake of its ligands by malignant cells are both strongly regulated by MT1-MMP. Because LRP is essential for the clearance of multiple ligands, these findings have important implications for many pathophysiological processes including the pericellular proteolysis in neoplastic cells as well as the fate of the soluble matrix-degrading proteases such as MMP-2. MT1-MMP is a key protease in cell invasion and a physiological activator of MMP-2. Cellular LRP consists of a non-covalently associated 515-kDa extracellular alpha-chain (LRP-515) and an 85-kDa membrane-spanning beta-chain, and plays a dual role as a multifunctional endocytic receptor and a signaling molecule. Through the capture and uptake of several soluble proteases, LRP is involved in the regulation of matrix proteolysis. LRP-515 associates with the MT1-MMP catalytic domain and is highly susceptible to MT1-MMP proteolysis in vitro. Similar to MT1-MMP, the metalloproteinases MT2-MMP, MT3-MMP and MT4-MMP also degrade LRP. The N-terminal and C-terminal parts of the LRP-515 subunit are resistant and susceptible, respectively, to MT1-MMP proteolysis. In cells co-expressing LRP and MT1-MMP, the proteolytically competent protease decreases the levels of cellular LRP and releases its N-terminal portion in the extracellular milieu while the catalytically inert protease co-precipitates with LRP. These events implicate MT1-MMP, not only in the activation of MMP-2, but also in the mechanisms that control the subsequent fate of MMP-2 in cells and tissues.     

Extracellular matrix formation by epithelial cells from human polycystic kidney cysts in culture.

Cells from the cysts of patients with autosomal dominant polycystic kidney disease (PKD) were grown in vitro under standard conditions without the aid of collagen-pretreated surfaces, and both the synthesis and composition of the extracellular matrix were investigated. At confluence, PKD cells presented the typical features of epithelial cells, but showed a different collagen composition from fibroblasts. Compared with normal tubular epithelia (NTE), PKD monolayers produced an excess of extracellular matrix, which accounted for 30% of the total incorporation of [3H] proline, although this value was considerably lower (by a factor of 10) in the case of NTE. Immunohistochemical and electrophoretic techniques revealed a complex collagen composition in the extracellular matrix which included [alpha (III)]3 and collagen IV. However, part of the collagen components remained unidentified in spite of the fact that they exhibited a typical M(r) of alpha 1(I) and alpha 2(I) in the presence of urea. Immunoprecipitation with monospecific antibodies and Northern blotting with specific probes failed to recognize alpha 1(I) and alpha 2(I), but demonstrated their presence in fibroblasts. Purification and cyanogen bromide digestion demonstrated a strong interhomology in fingerprint peptide composition among the uncharacterized collagens synthesized by PKD cells, thus suggesting a common identity. These observations document a markedly augmented production of extracellular matrix by PKD cultured cells in vitro, and show the presence of collagens which do not share homologies with the major collagen molecules. A better characterization of extracellular matrix composition is central to any comprehension of the cytogenetic mechanisms in vivo.     

The major low molecular weight apolipoprotein from normal and hyperlipidemia atherosclerosis-prone (LAP) Japanese quail

The low molecular weight (LMW) apolipoprotein of apo C plays an important role in the metabolism of triglyceride-rich lipoproteins. This study aimed at a characterization of the major LMW apolipoproteins from normal quail strain, and also from LAP (hyperlipidemia atherosclerosis-prone) strain to identify its genetic disorder. The major LMW apoprotein cDNA clone from normal quail comprised of approximately 500 bp, and encoded polypeptide of 78 amino acid residues containing 57 amino acids as a mature apolipoprotein. Although the quail LMW apoprotein showed a low homology to either apo C-I, C-II, or C-III of other animals, it retained a well-developed amphipathic alpha-helix structure. There was no difference in the deduced primary structure of the quail LMW apoprotein between LAP and normal strain. An analysis of the mRNA expression showed that the quail LMW apoprotein was only expressed in the liver of both LAP and normal Japanese quail. No difference was noted in the hepatic expression of the quail LMW apoprotein mRNA between normal and LAP strains with neither normal nor atherogenic dietary conditions. The structure and expression of the major LMW apoprotein thus had no relevance to higher susceptibility of LAP strain to the experimental atherosclerosis.     

Matrix metalloproteinase 3 (stromelysin) activates the precursor for the human matrix metalloproteinase 9.

Matrix metalloproteinase 9 (MMP-9), also known as 92-kDa gelatinase/type IV collagenase, is secreted from neutrophils, macrophages, and a number of transformed cells in zymogen form. Here we report that matrix metalloproteinase 3 (MMP-3/stromelysin) is an activator of the precursor of matrix metalloproteinase 9 (proMMP-9). MMP-3 initially cleaves proMMP-9 at the Glu40-Met41 bond located in the middle of the propeptide to generate an 86-kDa intermediate. Cleavage of this bond triggers a change in proMMP-9 that renders the Arg87-Phe88 bond susceptible to the second cleavage by MMP-3, resulting in conversion to an 82-kDa form. alpha 2-Macroglobulin binding studies of partially activated MMP-9 demonstrate that the 82-kDa species is proteolytically active, but not the initial intermediate of 86 kDa. This stepwise activation mechanism of proMMP-9 is analogous to those of other members of the MMP family, but the action of MMP-3 on proMMP-9 is the first example of zymogen activation that can be triggered by another member of the MMP family. The results imply that MMP-3 may be an effective activator of proMMP-9 in vivo.     

Steady-state kinetics of low molecular weight (type-II) NADH dehydrogenase.

(1) The steady-state kinetics of the NADH dehydrogenase activity of Type-II (low molecular weight) NADH dehydrogenase with the acceptors ferricyanide, cytochrome c and 2,6-dichloroindophenol are consistent with the simultaneous operation of an ordered and a ping-pong mechanism. Thus, depending on the acceptor concentration, the reduced enzyme is preferentially oxidized before or after NAD+ disociates from it. (2) The acceptors are able to oxidize the reduced enzyme and its NAD+ complex equally well. In contrast to the kinetics of the Type-I (high molecular weight) enzyme, double substrate inhibition is not found, implying that the site of oxidation of the reduced enzyme by acceptors and the NADH-binding site are remote. (3) With the indophenol, in the concentration range measured, the ordered mechanism is mainly operative. At infinite NADH and acceptor concentrations the rate constant of the reduction of enzyme by bound NADH is measured. (4) With ferricyanide and cytochrome c, in the concentration range measured, erroneous conclusions may be drawn from extrapolations owing to the fact that extrapolated lines in double-reciprocal plots of turnover number against acceptor concentration, at different NADH concentrations, intersect in the third quadrant. A method is described that allows the extrapolation of these data to zero acceptor concentrations. (5) The relation between activity and NADH concentration is sigmoidal (h = 2.0) with ferricyanide or cytochrome c as acceptor, but hyperbolic with 2,6-dichloroindophenol. The latter is also an inhibitor, competitive with respect to NADH. It is concluded that this two-electron acceptor, like ubiquinone, acts as an allosteric effector. (6) Type II is isolated from Type I without gross changes in tertiary structure, as judged by the unaltered rate constants of dissociation of NADH (k-1) and NAD+ (k4) and association of NADH (k1). (7) Type II differs from Type I in two respects, (a) The accessibility of the acceptors is greater by at least two orders of magnitude (k3). (b) The redox potential of the prosthetic group FMN is 120 mV less, as judged by a drop in the value of k2 by four orders of magnitude. It is suggested that one or more of the iron-sulphur proteins present in Type-I but lacking in Type-II dehydrogenase functions as an effector, regulating the redox potential of the FMN.     

Extracellular matrix formation by epithelial cells from human polycystic kidney cysts in culture

Cells from the cysts of patients with autosomal dominant polycystic kidney disease (PKD) were grown in vitro under standard conditions without the aid of collagen-pretreated surfaces, and both the synthesis and composition of the extracellular matrix were investigated. At confluence, PKD cells presented the typical features of epithelial cells, but showed a different collagen composition from fibroblasts. Compared with normal tubular epithelia (NTE), PKD monolayers produced an excess of extracellular matrix, which accounted for 30% of the total incorporation of [³H] proline, although this value was considerably lower (by a factor of 10) in the case of NTE. Immunohistochemical and electrophoretic techniques revealed a complex collagen composition in the extracellular matrix which included [α(III)]₃ and collagen IV. However, part of the collagen components remained unidentified in spite of the fact that they exhibited a typical M_r of α₁(I) and α₂(I) in the presence of urea. Immunoprecipitation with monospecific antibodies and Northern blotting with specific probes failed to recognize α₁(I) and α₂(I), but demonstrated their presence in fibroblasts. Purification and cyanogen bromide digestion demonstrated a strong interhomology in fingerprint peptide composition among the uncharacterized collagens synthesized by PKD cells, thus suggesting a common identity. These observations document a markedly augmented production of extracellular matrix by PKD cultured cells in vitro, and show the presence of collagens which do not share homologies with the major collagen molecules. A better characterization of extracellular matrix composition is central to any comprehension of the cystogenetic mechanisms in vivo.     

The complete amino acid sequence of low molecular mass urokinase from human urine

The sequence of all 253 amino acids of the heavy (B-) chain of human urinary urokinase was determined. The fragmentation strategy employed included cyanogen bromide cleavage of S-carboxymethylated B-chain at Met and/or Trp residues, cleavage of acid-labile Asp-Pro bonds, and the use of the specific endoproteinases Lys-C and Arg-C for generation of overlapping fragments. For sequence determination automated solid- or liquid-phase techniques of Edman degradation were used. The amino acid sequence obtained substantiates the serine protease character of the B-chain of urokinase: a considerable homology with other serine proteinases, especially with the B-chain of human plasmin, was proved. The pertinent active site amino acids were localized: His-46, Asp-97, and Ser-198. A carbohydrate side chain, containing at least 4 glucosamine and 2 galactosamine residues, was demonstrated to be fixed at asparagine in position 144. The sequence data presented, together with the sequence of the second (A1-) chain of low molecular mass urokinase which was reported by us in an earlier communication, complete the knowledge of the whole primary structure of an active form of human urinary urokinase.     

Membrane type 1 matrix metalloproteinase (MT1-MMP/MMP-14) cleaves and releases a 22-kDa extracellular matrix metalloproteinase inducer (EMMPRIN) fragment from tumor cells

Proteolytic shedding is an important step in the functional down-regulation and turnover of most membrane proteins at the cell surface. Extracellular matrix metalloproteinase inducer (EMMPRIN) is a multifunctional glycoprotein that has two Ig-like domains in its extracellular portion and functions in cell adhesion as an inducer of matrix metalloproteinase (MMP) expression in surrounding cells. Although the shedding of EMMPRIN is reportedly because of cleavage by metalloproteinases, the responsible proteases, cleavage sites, and stimulants are not yet known. In this study, we found that human tumor HT1080 and A431 cells shed a 22-kDa EMMPRIN fragment into the culture medium. The shedding was enhanced by phorbol 12-myristate 13-acetate and inhibited by TIMP-2 but not by TIMP-1, suggesting the involvement of membrane-type MMPs (MT-MMPs). Indeed, down-regulation of the MT1-MMP expression in A431 cells using small interfering RNA inhibited the shedding. The 22-kDa fragment was purified, and the C-terminal amino acid was determined. A synthetic peptide spanning the cutting site was cleaved by MT1-MMP in vitro. The cleavage site is located in the linker region connecting the two Ig-like domains. The N-terminal Ig-like domain is important for the MMP inducing activity of EMMPRIN and for cell-cell interactions, presumably through its ability to engage in homophilic interactions, and the 22-kDa fragment retained the ability to augment MMP-2 expression in human fibroblasts. Thus, the MT1-MMP-dependent cleavage eliminates the functional N-terminal domain of EMMPRIN from the cell surface, which is expected to down-regulate its function. At the same time, the released 22-kDa fragment may mediate the expression of MMPs in tumor tissues.     

In vivo footprinting of a low molecular weight glutenin gene (LMWG-1D1) in wheat endosperm.

The quality of the wheat grain is determined by the quantity and composition of storage proteins (prolamins) which are synthesized exclusively in endosperm tissue. We are investigating the mechanisms underlying the regulation of expression of a prolamin gene, the low molecular weight glutenin gene LMWG-1D1. The LMWG-1D1 promoter contains the endosperm box, a sequence motif highly conserved in the promoter region of a large number of storage protein genes, which is thought to confer endosperm-specific expression of prolamin genes. Here we show by in vivo DMS footprinting of wheat endosperm tissue that the endosperm box becomes occupied by putative trans-acting factors during grain ripening. During early stages of development the endosperm motif within the 5' half of the endosperm box becomes occupied first, followed by binding of a second activity to a GCN4/jun-like motif in the 3' half just prior to the stage of maximum gene expression. Occupancy of the endosperm box is highly tissue-specific: no protection was observed in husk and leaf tissues. Several binding activities were identified in vitro from nuclear protein extracts of wheat endosperm which bind specifically to the endosperm and GCN4/jun motifs identified by in vivo footprinting.     

An alternative processing of integrin alpha(v) subunit in tumor cells by membrane type-1 matrix metalloproteinase.

Membrane type-1 matrix metalloproteinase (MT1-MMP) and alpha(v)beta(3) integrin are both essential to cell invasion. Maturation of integrin pro-alpha(v)chain (pro-alpha(v)) involves its cleavage by proprotein convertases (PC) to form the disulfide-bonded 125-kDa heavy and 25-kDa light alpha chains. Our report presents evidence of an alternative pathway of pro-alpha(v) processing involving MT1-MMP. In breast carcinoma MCF7 cells deficient in MT1-MMP, pro-alpha(v) is processed by a conventional furin-like PC, and the mature alpha(v) integrin subunit is represented by the 125-kDa heavy chain and the 25-kDa light chain commencing from the N-terminal Asp(891). In contrast, in cells co-expressing alpha(v)beta(3) and MT1-MMP, MT1-MMP functions as an integrin convertase. MT1-MMP specifically cleaves pro-alpha(v), generating a 115-kDa heavy chain with the truncated C terminus and a 25-kDa light chain commencing from the N-terminal Leu(892). PC-cleavable alpha(3) and alpha(5) but not the PC-resistant alpha(2) integrin subunit are also susceptible to MT1-MMP cleavage. These novel mechanisms involved in the processing of integrin alpha subunits underscore the significance and complexity of interactions between MT1-MMP and adhesion receptors and suggest that regulation of integrin functionality may be an important role of MT1-MMP in migrating tumor cells.     

Purification and immunochemical properties of human low molecular weight kininogen.

A low molecular weight (LMW) kininogen was isolated from pooled human serum by chromatography on DEAE-Sephadex A-50, CM-Sephadex C-50, Sephadex G-150, and Sephadex G-100. It was shown to be homogeneous by ultracentrifugation, polyacrylamide gel electrophoresis, and immunoelectrophoresis. The sedimentation coefficient, S020,W, of purified LMW kininogen was 3.85 s, and its molecular weight was determined to be 78,000 by Sephadex G-100 gel-filtration. The LMW kininogen contained 79.3% protein, 8.0% hexose, 3.9% hexosamine, and 4.9% sialic acid. In order to determine the immunochemical properties of LMW kininogen, specific antiserum was prepared in rabbits. The antigenic determinant of LMW kininogen was not related to the sialic acid and kinin moieties in the kininogen molecule, but could not be distinguished from that of high molecular weight (HMW) kininogen. In the quantitative single radial immunodiffusion test, a sialic acid-free LMW kininogen reacted to a greater extent with the antiserum than the native LMW kininogen. The kininogen level in human serum was estimated by single radial immunodiffusion. The antiserum cross-reacted with monkey serum, but not with sera from dogs, rats, and mice, horses, pigs, guinea pigs, oxen, and rabbits.