Chemical modification of tissue inhibitor of metalloproteinases-1 and its inactivation by diethyl pyrocarbonate.

Tissue inhibitor of metalloproteinases-1 (TIMP-1) was treated with a range of chemical modification reagents in order to identify amino acid residues essential for inhibitory activity. Diethyl pyrocarbonate (DEPC) was found to be a potent inactivator at low reagent/TIMP molar concentrations. The extent of modification at 50% inactivation was determined as 1.5 sites/molecule. The DEPC-modified inhibitor did not form stable complexes with stromelysin, but was shown to retain native structure as judged by conformational stability to denaturation by guanidine hydrochloride. Peptide mapping experiments were used to find the sites of DEPC incorporation within the primary structure of TIMP and three residues were identified (His-95, His-144 and His-164). Mutant TIMPs in which histidine residues have been substituted or deleted retain inhibitory activity and were found to be equally as sensitive to DEPC inactivation as the wild-type. No new sites of DEPC modification in the mutant proteins were detected. The possible contribution made by His residues 95, 144 and 164 to the inhibitory activity of TIMP is discussed.

Structural analysis of tissue inhibitor of metalloproteinases-1 (TIMP-1) by tryptic peptide mapping.

Tryptic digests of recombinant TIMP-1 have been resolved on reverse-phase HPLC and the major peaks identified by N-terminal sequencing. This procedure accounted for the entire molecule, except two short peptides of 2 and 4 amino acids in length. The peptide map was used to (i), characterize an insoluble 'core' peptide seen on digestion of TIMP-1 in non-reducing conditions; (ii), confirm the structure of delta 127-184TIMP-1, a recently described truncated form of the TIMP-1 molecule; (iii), identify exposed regions of the intact and truncated TIMP-1 molecules by measuring the rate of tryptic peptide release and (iv), locate sites of aberrant proteolysis seen when recombinant human TIMP-1 was purified at large scale.

A single amino acid substitution abolishes the heterogeneity of chimeric mouse/human (IgG4) antibody.

Human immunoglobulin G4 (IgG4) exists in two molecular forms due to the heterogeneity of the inter-heavy chain disulphide bridges in the hinge region in a proportion of secreted human IgG4. This heterogeneity is only revealed under denaturing, non-reducing conditions in which an HL "half antibody" is detected, a phenomenon not seen in other human IgG isotypes. In native conditions noncovalent interactions hold the antibody together as the H2L2 tetramer. Analysis of the hinge sequences of human IgG heavy chains suggested that the presence of serine at residue 241 might be the cause of this heterogeneity. We therefore changed the serine at 241 to proline (found at that position in IgG1 and IgG2) in a mouse/human chimeric heavy chain. This single residue substitution leads to the production of a homogeneous antibody. Further, the variant IgG4 has significantly extended serum half-life and shows an improved tissue distribution compared to the original chimeric IgG4.

The matrix metalloproteinases and their natural inhibitors: prospects for treating degenerative tissue diseases.

Uncontrolled matrix metalloproteinase activity is thought to be a cause of the tissue damage observed in many disease processes. None of the drugs currently in use can prevent tissue destruction, and strategies for the development of synthetic inhibitors have been hampered by a poor understanding of the biochemistry of matrix metalloproteinases. Recent cDNA cloning efforts and characterization of recombinant human matrix metalloproteinases have permitted structure-function analysis of the enzymes and their inhibitors. Progress in this area should help indicate a route to rational strategies for designing lead therapeutic compounds.

Expression, purification and characterization of a mouse-human chimeric antibody and chimeric Fab' fragment.

B72.3 is a mouse monoclonal antibody against a tumour-associated antigen, TAG72, which recognizes breast, ovarian and colorectal tumour tissue. A mouse-human chimeric version of B72.3 has been expressed in Chinese-hamster ovary cells. This molecule has the binding specificity of B72.3 and constant regions from human IgG4. The chimeric B72.3 assembles to intact IgG and recognizes TAG72 as well as B72.3 in competitive binding assays. A proportion of the chimeric B72.3 (approx. 10%) does not form inter-heavy-chain disulphide bonds but still assembles into the IgG tetramer. This appears to be a general property of human IgG4 molecules. Co-expression of the chimeric light chain with a chimeric Fd' gene resulted in the expression of functional Fab'. Very little F(ab')2 is produced, although the Fab' can be oxidized to the dimeric F(ab')2 in vitro. The production of Fab' and F(ab')2 by this method is an attractive alternative to proteolytic digestion of IgG. The ability to produce these molecules in large quantities will allow the production and testing of a range of anti-tumour antibody and antibody fragment conjugates.

Binding of latent and high Mr active forms of stromelysin to collagen is mediated by the C-terminal domain.

A specific high-titre polyclonal antiserum to recombinant human prostromelysin was raised in a sheep and shown by immunoblotting to detect latent prostromelysin, high and low Mr active forms and the C-terminal domain. This antiserum was used to demonstrate by indirect immunofluorescence that latent and active high Mr prostromelysin bind to reconstituted collagen fibrils, and to other extracellular matrix components in tissues ex vivo but that active low Mr stromelysin does not. Isolation of the C-terminal domain was carried out to demonstrate that stromelysin binding was through this domain. By use of an antiserum to the tissue inhibitor of metalloproteinases (TIMP) it was shown that TIMP is unable to bind to reconstituted collagen fibrils. TIMP, however, will bind when active high Mr stromelysin is present but not if latent prostromelysin is bound. We conclude that stromelysin has different binding specificities from those previously documented for collagenase; only active collagenase binds to reconstituted collagen fibrils. However, TIMP binds to the active forms of both stromelysin and collagenase when these are bound to the collagen fibrils. These results have important implications for the interpretation of immunolocalization data in establishing the roles of metalloproteinases and their inhibitors in vivo.

Purification of recombinant human prostromelysin. Studies on heat activation to give high-Mr and low-Mr active forms, and a comparison of recombinant with natural stromelysin activities.

Recombinant human prostromelysin was purified in a single step using Procion Red-Sepharose chromatography. The purified prostromelysin was self-activated to high-Mr (45,000) and low-Mr (28,000) forms by incubation at 55 degrees C without the addition of extraneous activators. The two forms of stromelysin were subsequently separated, again using Procion Red-Sepharose. Both of the heat-activated recombinant forms demonstrated similar specific activities (for the macromolecular substrates casein, gelatin, elastin, proteoglycan and type IV collagen) when compared with either heat- or trypsin-activated natural stromelysin. The heat-activated recombinant stromelysins both showed similar abilities to potentiate activation of human procollagenase when compared with trypsin-activated natural stromelysin.

Purification of a hybrid plasminogen activator protein.

Recombinant DNA technology has been employed to produce a hybrid gene in which the kringle and serine protease domains of tissue plasminogen activator are linked to the heavy-chain Fd region of a fibrin-specific antibody. The hybrid gene is co-expressed with antibody light chains. This communication describes a purification procedure for the hybrid protein, involving affinity and ion-exchange chromatography. The purified hybrid protein has been used in vivo and in vitro clot lysis experiments and has been shown to be effective at clot dissolution.

Disulphide bond assignment in human tissue inhibitor of metalloproteinases (TIMP).

Disulphide bonds in human recombinant tissue inhibitor of metalloproteinases (TIMP) were assigned by resolving proteolytic digests of TIMP on reverse-phase h.p.l.c. and sequencing those peaks judged to contain disulphide bonds by virtue of a change in retention time on reduction. This procedure allowed the direct assignment of Cys-145-Cys-166 and the isolation of two other peptides containing two disulphide bonds each. Further peptide cleavage in conjunction with fast-atom-bombardment m.s. analysis permitted the assignments Cys-1-Cys-70, Cys-3-Cys-99, Cys-13-Cys-124 and Cys-127-Cys-174 from these peptides. The sixth bond Cys-132-Cys-137 was assigned by inference, as the native protein has no detectable free thiol groups.

Dye-ligand chromatography.

The affinity of two kinases for Blue Dextran was discovered in 1968 during gel permeation chromatography (1) and agarose electrophoresis (2). The observed interaction was mediated by the chromophore of Blue Dextran, Cibacron Blue F3G-A (3), which was postulated to bind to the supersecondary structure of certain enzymes known as the dinucleotide fold (4). Immobilized Cibacron Blue F3G-A replaced more expensive biological group-specific ligands in the affinity chromatographic purification of a wide variety of nucleotide-binding enzymes (5). The adsorbent was easier to synthesize, biochemically more stable, and had much higher capacities than the traditional nucleotide-based affinity matrices, making it more suitable for large-scale purifications.

Molecular cloning of a human gastric lipase and expression of the enzyme in yeast.

The molecular cloning of a cDNA coding for human gastric lipase and its expression in yeast is described. A lipase present in human gastric aspirates was purified and its N-terminal amino-acid sequence was determined. This was found to be homologous with the N-terminal sequence of rat lingual lipase. A cDNA library was constructed from mRNA isolated from human stomach tissue and probed with cloned rat lingual lipase DNA. One clone, pGL17, consisting of approximately 1450 base-pairs, contained the entire coding sequence for a human gastric lipase. The amino-acid sequence from the isolated protein and the DNA sequence obtained from the cloned gene indicated that human gastric lipase consists of a 379 amino acid polypeptide with an unglycosylated Mr of 43,162. Human gastric lipase and rat lingual lipase amino-acid sequences were closely homologous but were unrelated to porcine pancreatic lipase apart from a 6 amino-acid sequence around the essential Ser-152 of porcine pancreatic lipase. A yeast expression plasmid containing the phosphoglycerate kinase promoter and terminator sequences together with the human gastric lipase gene was constructed. Yeast transformed with this vector synthesised the lipolytically active enzyme.

Molecular cloning and nucleotide sequence of rat lingual lipase cDNA.

Purified rat lingual lipase (EC3113), a glycoprotein of approximate molecular weight 52,000, was used to generate polyclonal antibodies which were able to recognise the denatured and deglycosylated enzyme. These immunoglobulins were used to screen a cDNA library prepared from mRNA isolated from the serous glands of rat tongue cloned in E. coli expression vectors. An almost full length cDNA clone was isolated and the nucleotide and predicted amino acid sequence obtained. Comparison with the N-terminal amino acid sequence of the purified enzyme confirmed the identity of the cDNA and indicated that there was a hydrophobic signal sequence of 18 residues. The amino acid sequence of mature rat lingual lipase consists of 377 residues and shares little homology with porcine pancreatic lipase apart from a short region containing a serine residue at an analogous position to the ser 152 of the porcine enzyme.

Cooperative interactions in neurophysin-neuropeptide hormone complexes. Analytical affinity chromatography of native and covalently-modified neurophysins.

The structural interdependence between neurophysin (NP) self-association and ligand binding surfaces has been studied by analytical affinity chromatography of several NP sequence variants and derivatives on Met-Tyr-Phe-aminobutyl-agarose and bovine NP-II Sepharose. Elutions of radiolabeled NP's from both matrices show that hybrid dimers can form between major bovine NP's (I and II, or VLDV- and MSEL-NP's, respectively), as well as between human and bovine NP's, with affinities close to that for homologous dimer formation. Such evidence supports the view that the region of NP involved in NP-NP contact is composed primarily of conserved structural elements of the protein. NP antibodies which recognize surfaces close to or in the NP-NP contact region have been detected by their effects on bovine NP-II elution on NP-II Sepharose. Elutions of [3-nitro-Tyr 49] BNP-II from Met-Tyr-Phe-aminobutyl-agarose showed that nitration has little effect on the chromatographic properties of NP-II. This evidence substantiates previous arguments (Angal, S. & Chaiken, I.M. (1982) Biochemistry 21, 1574-1580) that the chromatographic behavior of native NP's on the affinity matrices is an expression of the interdependence of NP self-association and ligand binding surfaces and not due to bivalent peptide binding by NP monomer. The affinity chromatographic properties of NP derivatives, including bovine NP-II photolabeled in the ligand binding site and tryptic fragments of bovine NP-I (NP-I-(9-93) and [des 19-20] NP-I-(9-93)), support the view that the surfaces for ligand binding and NP-NP contact are conformationally linked. The data argue that conformational changes that ensue upon noncovalent ligand binding and lead to enhanced NP self-association cannot occur favorably with the protein modified by either covalent ligand attachment or limited amino-terminal proteolysis.

Synthesis of calf prochymosin (prorennin) in Escherichia coli.

A gene for calf prochymosin (prorennin) has been reconstructed from chemically synthesized oligodeoxyribonucleotides and cloned DNA copies of preprochymosin mRNA. This gene has been inserted into a bacterial expression plasmid containing the Escherichia coli tryptophan promoter and a bacterial ribosome binding site. Induction of transcription from the tryptophan promoter results in prochymosin synthesis at a level of up to 5% of total protein. The enzyme has been purified from bacteria by extraction with urea and chromatography on DEAE-cellulose and converted to enzymatically active chymosin by acidification and neutralization. Bacterially produced chymosin is as effective in clotting milk as the natural enzyme isolated from calf stomach.

Effects of limited tryptic proteolysis of bovine neurophysins on molecular properties of hormone binding, self-association, and antigenicity.

Limited tryptic fragmentation of disulfide-intact bovine neurophysins I and II (NP-I and -II, respectively) has been found to cause selective disruption of both hormone binding and neurophysin self-association. Loss of binding interactions, measured as a loss of ability to stimulate retardation of 125I-labeled neurophysin on Met-Tyr-Phe-amino-butylaminoagarose, is complete within 3 h at 37 degrees C. Reverse-phase high-performance liquid chromatography (HPLC) analysis of tryptic digests of neurophysin I allows detection of two major protein products and the peptide fragment 1-8. Release of the latter N-terminal piece occurs at about the same rate as loss of binding interactions. Reverse-phase HPLC elution behavior before and after performic acid oxidation and amino acid composition of the protein products led to their identification as NP-I-(9-93) (the 9-93 sequence) and [des-19,20]NP-I-(9-93) (the 9-93 sequence with the dipeptide 19-20 missing) for the more rapidly and more slowly formed species, respectively. NP-I-(9-93), unlike intact neurophysin I, is not retarded strongly by either Met-Tyr-Phe-amino-butylaminoagarose or neurophysin II-Sepharose. In contrast, both NP-I-(9-93) and [des-19,20]NP-I-(9-93) are equally as effective as intact NP-I in binding neurophysin I antibodies. The role of amino-terminal residues in promoting hormone binding, self-association, and antigenic recognition interactions is considered.