Kjeld Norris

2-(oxalylamino)-benzoic acid is a general, competitive inhibitor of protein-tyrosine phosphatases.

Protein-tyrosine phosphatases (PTPs) are critically involved in regulation of signal transduction processes. Members of this class of enzymes are considered attractive therapeutic targets in several disease states, e.g. diabetes, cancer, and inflammation. However, most reported PTP inhibitors have been phosphorus-containing compounds, tight binding inhibitors, and/or inhibitors that covalently modify the enzymes. We therefore embarked on identifying a general, reversible, competitive PTP inhibitor that could be used as a common scaffold for lead optimization for specific PTPs. We here report the identification of 2-(oxalylamino)-benzoic acid (OBA) as a classical competitive inhibitor of several PTPs. X-ray crystallography of PTP1B complexed with OBA and related non-phosphate low molecular weight derivatives reveals that the binding mode of these molecules to a large extent mimics that of the natural substrate including hydrogen bonding to the PTP signature motif. In addition, binding of OBA to the active site of PTP1B creates a unique arrangement involving Asp181, Lys120, and Tyr46. PTP inhibitors are essential tools in elucidating the biological function of specific PTPs and they may eventually be developed into selective drug candidates. The unique enzyme kinetic features and the low molecular weight of OBA makes it an ideal starting point for further optimization.

Characterization of recombinant human HBP/CAP37/azurocidin, a pleiotropic mediator of inflammation-enhancing LPS-induced cytokine release from monocytes

Neutrophil‐derived heparin‐binding protein (HBP) is a strong chemoattractant for monocytes. We report here for the first time the expression of recombinant HBP. A baculovirus containing the human HBP cDNA mediated in insect cells the secretion of a 7‐residue N‐terminally extended HBP form (pro‐HBP). Deletion of the pro‐peptide‐encoding cDNA sequence resulted in correctly processed HBP at the N‐terminus. Electrospray mass spectrum analysis of recombinant HBP yielded a molecular weight of 27.237 ± 3 amu. Consistent with this mass is a HBP form of 225 amino acids (mature part +3 amino acid C‐terminal extension). The biological activity of recombinant HBP was confirmed by its chemotactic action towards monocytes. Furthermore, we have shown that recombinant HBP stimulates in a dose‐dependent manner the lipopolysaccharide (LPS)‐induced cytokine release from human monocytes.

Purification and characterization of the trefoil peptide human spasmolytic polypeptide (hSP) produced in yeast

Recombinant human spasmolytic polypeptide (r‐hSP) has been produced in relatively large amounts in Saccharomyces cerevisiae. The two intronless trefoil domains of the hSP‐DNA were cloned separately by PCR from human genomic DNA, and the remaining parts of the gene synthezised. Recombinant plasmids were constructed to encode a fusion protein consisting of a hybrid leader sequence and the hSP sequence. The leader sequence serves to direct the fusion protein into the secretory pathway of the cell and to expose it to the Kex 2 processing enzyme system. The secreted r‐hSP was found in a glycosylated and an non‐glycosylated form. The two forms of r‐hSP were purified from the yeast fermentation broth by a combination of ion‐exchange chromatography and preparative HPLC. The overall yield from 8 litres of fermentation broth was 160 mg r‐hSP and 219 mg glycosylated r‐hSP corresponding to 50% and 34%, respectively. The structure of the r‐hSP and the glycosylated r‐hSP was determined by amino acid analysis and carbohydrate composition analysis as well as by peptide mapping, amino acid sequencing and mass spectrometric analysis.

EXPRESSION OF PROTEIN-TYROSINE PHOSPHATASES IN THE MAJOR INSULIN TARGET TISSUES

Protein‐tyrosine phosphatases (PTPs) are key regulators of the insulin receptor signal transduction pathway. We have performed a detailed analysis of PTP expression in the major human insulin target tissues or cells (liver, adipose tissue, skeletal muscle and endothelial cells). To obtain a representative picture, all tissues were analyzed by PCR using three different primer sets corresponding to conserved regions of known PTPs. A total of 24 different PTPs were identified. A multiprobe RNase protection assay was developed to obtain a semi‐quantitative measure of the expression levels of selected PTPs. Surprisingly, PTP‐LAR, previously suggested to be a major regulator of the insulin receptor tyrosine kinase, was expressed in extremely low levels in skeletal muscle, whereas the related receptor‐type PTP‐σ and PTP‐α were expressed in relatively high levels in all four tissues. The low levels of LAR PTP mRNA in skeletal muscle were further confirmed by Northern blot analysis.

The 1.6 A structure of Kunitz-type domain from the alpha 3 chain of human type VI collagen.

The C-terminal Kunitz-type domain from the alpha 3 chain of human type VI collagen (C5), a single 58 amino acid residue chain with three disulfide bridges, was cloned, expressed and crystallized in a monoclonic form, space group P2(1), with a = 25.7 A, b = 38.2 A, c = 28.8 A and beta = 109 degrees. The structure was resolved by molecular replacement, using Alzheimers protein precursor inhibitor and bovine pancreatic trypsin inhibitor three-dimensional structures as search models. The molecule with one sulfate ion and 43 associated water molecules was refined by XPLOR to an R-factor of 18.9% at 1.6 A. The molecule was not degraded by trypsin and did not inhibit trypsin or tested serine proteases. As opposed to the other Kunitz family members, C5 demonstrates left-handed chirality of the Cys14-Cys38 disulfide bond. Inversion of the Thr13 carbonyl and bulky side-chains at the interface with trypsin in a model of the C5-trypsin complex may explain the lack of inhibition of trypsin.

Expression, purification and characterization of a Kunitz-type protease inhibitor domain from human amyloid precursor protein homolog

The Kunitz‐type protease inhibitor domain from a recently identified homolog of the Alzheimer amyloid precursor protein (APPH KPI) was expressed in yeast, purified and characterized. Its inhibition profile towards several serine proteases was studied and compared to that of APP KPI, the Kunitz domain from the Alzheimer amyloid precursor protein. APPH KPI was shown to inhibit proteases with trypsin‐like specificity with an inhibitor profile resembling that of the APP KPI domain. The KPI domains from APP and APPH inhibited trypsin (K i = 0.02 nM), and plasma kallikrein (K i = 86 nM) with approximal equal affinity. In comparison to APP KPI (K i = 82 nM) the KPI domain of the homolog, APPH KPI, (K i = 8.8 nM) was a more potent inhibitor of glandular kallikrein. APPH KPI was a less potent inhibitor of chymotrypsin than APP KPI (K i = 78 nM as compared to K i = 6 nM), plasmin (K i = 81 nM as compared to 42 nM), and factor XIa (K i = 14 nM as compared to K i = 0.7 nM). The affinity of factor XIa for APPH KPI is sufficiently high to allow for an interaction in the blood. It is, however, well possible that the physiological protease ligand for the receptor‐like APPH protein has yet to be identified.

The secretion of glucagon by transformed yeast strains

Saccharomyces cerevisiae strains were transformed with plasmids coding for modified mating factor α1 leader sequences followed by glucagon. Glucagon‐containing peptides which were secreted into the fermentation broth were isolated and their amino acid sequences determined. The yeast strain transformed with the sequence coding for the complete mating factor α1 leader sequence preceding the glucagon gene (MT556) secreted glucagon plus glucagon extended at its N‐terminal by parts of the leader sequence. The yeast strain transformed with the sequence coding for a truncated mating factor α1 leader sequence before the glucagon gene (MT615) secreted glucagon. These observations suggest that S. cerevisiae is a suitable vehicle for the efficient expression of plasmids coding for polypeptides similar to glucagon (e.g. VIP, secretin, GIP).

1.2 Å Refinement of the Kunitz-Type Domain from the α3 Chain of Human Type VI Collagen

The recombinant Kunitz-type domain (C5) of human collagen alpha3(VI) chain was previously described at 1.6 A resolution at room temperature. By changing the crystallization conditions and using synchrotron radiation, we are able to record diffraction data to 1.2 A resolution for crystals of the same space group at 291 K. The protein-water-ion model has been refined anisotropically against these new data using the program SHELXL93; the results converged to an R factor of 15.0%, with all data between 7 and 1.2 A. The final electron-density map reveals a clear chain tracing with a few disordered residues and five residues out of 58 that present alternate conformations. The Cys14-Cys38 bond presents the less frequently observed left-hand conformation (chi1 = -60 degrees). The solvent molecules and a phosphate ion are well ordered with an average B of 38 A2. The high-resolution structure reveals the N and C termini which were missing from the 1.6 A structure.

Elucidation of the origin of multiple conformations of the human α3-chain type VI collagen C-terminal Kunitz domain: The reorientation of the Trp21 ring

SummaryThe human α3-chain type VI collagen C-terminal Kunitz domain fragment (α3(VI)) has been studied by two-dimensional 1H−1H and 1H−13C NMR spectroscopy at 303 K. It is shown that the secondary structure of the protein is strikingly similar to that of BPTI, and that a number of unusual Hα chemical shifts, which are highly conserved in Kunitz-domain proteins, are also observed for α3(VI). Further-more a series of exchange cross peaks observed in 1H−1H spectra shows that a large number of protons in the central β-sheet exist in two different chemical environments, corresponding to two unequally populated conformations that are slowly exchanging on the NMR time scale. Several protons, including Ser47(53) Hα, Arg32(38) Hγ2, and Gln48(54) Hβ2, all located in the vicinity of the Trp21(27) ring in the crystal structure of α3(VI) [Arnoux, B. et al. (1995) J. Mol. Biol., 246, 609–617], have very different chemical shifts in the two conformations, the most affected being Gln48(54) Hβ2 (Δδ=1.53 ppm), which is placed directly above the Trp21(27) ring in the crystal structure of α3(VI). It is concluded that the origin of the multiple conformations of the central β-sheet is a reorientation of the Trp21(27) ring. From the intensities of corresponding signals in the two conformations, the population of the minor conformation was found to be 6.4±0.2% of that of the major conformation, while a rate constant kM=1.01±0.05 s-1 for the major to minor interconversion was obtained from a series of NOESY spectra with different mixing times. In addition, it is shown that Cys14(20)-Cys38(44) disulfide bond isomerization, previously observed in BPTI [Otting, G. et al. (1993) Biochemistry, 32, 3570–3582], is also likely to occur in α3(VI).

Identifying the functional part of heparin-binding protein (HBP) as a monocyte stimulator and the novel role of monocytes as HBP producers.

Heparin-binding protein (HBP), an evolutionary ancient and biologically highly important molecule in inflammation, is an inactive serine protease due to mutations in the catalytic triad. The histidine (position 41) in the conserved sequence TAAHC is mutated to serine and this sequence (TAASC) plays a crucial role when HBP binds to monocytes. We synthesized a 20—44 HBP peptide, cyclicized by a sulphur bridge, which encompasses this amino acid and functions as full-length HBP. Using a human monocyte cell line, we have shown that lipopolysaccharide (LPS)-triggered secretion of IL-6 is enhanced up to 10-fold when full-length HBP or the peptide are present in low-to-moderate concentrations. A monoclonal antibody neutralizing HBP also neutralizes the peptide, indicating that the ligand for the HBP receptor is located near serine in position 41 on the HBP surface. A ‘back mutated’ 20—44 peptide (serine→histidine) has some, but not significant, stimulatory effect on monocytes. Normally, HBP production and release is ascribed to neutrophil granulocytes, but here we find that also monocytes secrete HBP when stimulated with LPS. Furthermore, a small amount of HBP can be demonstrated when monocytes are incubated in medium alone. Our efforts to identify a suggested HBP receptor on monocytes has failed so far.