Poul Baad Rasmussen

Establishment of two continuous T-cell strains from a single plaque of a patient with mycosis fungoides.

SummaryFrom a plaque biopsy of a patient with mycosis fungoides, two different continuous cell lines were established by including both IL-2 and IL-4 in culture medium. Both continuous cell lines appeared with characteristic chromosome markers after approximately 40 cell population doublings. The initial karyotype recognized in T cells from the skin biopsy was 46,XY and the karyotypes of the continuous cell strains were 46,XY, -18, + i(18q) and another with multiple chromosome aberrations as described in Sezary T-cell leukemia. Phenotyping with monoclonal antibodies and T-cell receptor analysis indicates that the latter cell strain represents a minority of T-cells in the plaque. Due to its many chromosomal aberrations it probably represents the malignant cell, which may be a central cell in the immune stimulation taking place in the skin.

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.

One-step purification and characterization of human pancreatic lipase expressed in insect cells

A cDNA clone encoding the sequence of human pancreatic lipase (HPL) was subcloned into the baculovirus transfer vector pVL1392 and used in co‐transfection of Spodoptera frugiperda (Sf9) insect cells with wild‐type Autographa californica nuclear polyhedrosis virus (AcNPV) DNA. A single recombinant protein (50 kDa) secreted by Sf9 cells was detectable in the culture medium 24 h post‐infection using both anti‐HPL polyclonal antibodies and potentiometric measurements of lipolytic activity. The expression level reached 40 mg/l of enzyme at 6 days. A single cation‐exchange chromatography was sufficient to obtain a highly pure recombinant HPL as demonstrated by N‐terminal sequencing, amino acid composition and carbohydrate analysis, as well as by mass spectrometry. These analyses revealed the production of mature protein with the correct processing of signal peptide and an homogenous glycosylation pattern. The kinetic properties of recombinant and native HPL were compared. Both enzymes showed similar profiles of interfacial activation, inhibition by bile salts and re‐activation by colipase.

Decreased agonist sensitivity of human GABAA receptors by an amino acid variant, isoleucine to valine, in the α1 subunit

Recombinant human GABA(A) receptors were investigated in vitro by coexpression of cDNAs coding for alpha1, beta2, and gamma2 subunits in the baculovirus/Sf-9 insect cell system. We report that a single amino acid exchange (isoleucine 121 to valine 121) in the N-terminal, extracellular part of the alpha1 subunit induces a marked decrease in agonist GABA(A) receptor ligand sensitivity. The potency of muscimol and GABA to inhibit the binding of the GABA(A) receptor antagonist [3H]SR 95531 (2-(3-carboxypropyl)-3-amino-6-(4-methoxyphenyl)pyridazinium bromide) was higher in receptor complexes of alpha1(ile 121) beta2gamma2 than in those of alpha1(val 121) beta2gamma2 (IC50 values were 32-fold and 26-fold lower for muscimol and GABA, respectively). The apparent affinity of the GABA(A) receptor antagonist bicuculline methiodide to inhibit the binding of [3H]SR 95531 did not differ between the two receptor complex variants. Electrophysiological measurements of GABA induced whole-cell Cl- currents showed a ten-fold decrease in the GABA(A) receptor sensitivity of alpha1 (val 121) beta2gamma2 as compared to alpha1(ile 121) beta2gamma2 receptor complexes. Thus, a relatively small change in the primary structure of the alpha1 subunit leads to a decrease selective for GABA(A) receptor sensitivity to agonist ligands, since no changes were observed in a GABA(A) receptor antagonist affinity and benzodiazepine receptor binding.

CHARACTERIZATION OF THE BIOSYNTHESIS, PROCESSING, AND SORTING OF HUMAN HBP/CAP37/AZUROCIDIN

Azurocidin is a multifunctional endotoxin‐ binding serine protease homolog synthesized during the promyelocytic stage of neutrophil development. To characterize the biosynthesis and processing of azurocidin, cDNA encoding human preproazurocidin was stably transfected to the rat basophilic leukemia cell line RBL‐1 and the murine myeloblast‐like cell line 32D cl3; cell lines previously utilized to study the related proteins cathepsin G and proteinase 3. After 30 min of pulse radiolabeling, two forms of newly synthesized proazurocidin (34.5 and 37 kDa), differing in carbohydrate content but with protein cores of identical sizes, were recognized. With time, the 34.5‐kDa form disappeared, while the 37‐kDa form was further processed proteolytically, as judged by digestion with N‐glycosidase F. Conversion of high‐mannose oligosaccharides into complex forms was shown by acquisition of complete resistance to endoglycosidase H. Radiosequence analysis demonstrated that the amino‐terminal seven amino acid propeptide of proazurocidin was removed in a stepwise manner during processing; initial removal of five amino acids was followed by cleavage of a dipeptide. Presence of the protease inhibitors Gly‐Phe‐diazomethyl ketone, bestatin, or leupeptin inhibited only the cleavage of the dipeptide, thus indicating the involvement of at least two amino‐ terminal processing enzymes. Translocation of azurocidin to granules was shown by subcellular fractionation. Similar results, with efficient biosynthesis, processing, and targeting to granules in both cell lines, were obtained with a mutant form of human preproazurocidin lacking the amino‐terminal heptapropeptide. In conclusion, this investigation is an important addition to our previous studies on related azurophil granule proteins, and provides novel information concerning the biosynthesis and distinctive amino‐terminal processing of human azurocidin. J. Leukoc. Biol. 66: 634–643; 1999.

Two mutants of human heparin binding protein (CAP37): Toward the understanding of the nature of lipid A/LPS and BPTI binding†

Heparin binding protein (HBP) is an inactive serine protease homologue with important implications in host defense during infections and inflammations. Two mutants of human HBP, [R23S,F25E]HBP and [G175Q]HBP, have been produced to investigate structure‐function relationships of residues in the putative lipid A/lipopolysaccharide (LPS) binding site and BPTI (bovine pancreatic trypsin inhibitor) binding site. The X‐ray structures have been determined at 1.9 Å resolution for [G175Q]HBP and at 2.5 Å resolution for the [R23S,F25E]HBP mutant, and the structures have been fully refined to R‐factors of 18.2% and 20.7%, respectively. The G175Q mutation does not alter the overall structure of the protein, but the ability to bind BPTI has been eliminated, and the mutant mediates only a limited stimulation of the LPS‐induced cytokine release from human monocytes. The lipid A/LPS binding property of [G175Q]HBP is comparable with that of native HBP. The R23S,F25E mutations do not affect the binding of lipid A/LPS and BPTI or the LPS‐induced cytokine release from human monocytes. This shows that two diverse ligands, lipid A/LPS and BPTI, do not share binding sites. Previously, there was convincing evidence for the proposed lipid A/LPS binding site of HBP. Unexpectedly, the extensive structural changes introduced by mutation of Arg23 and Phe25 do not affect the binding of lipid A/LPS, indicating that another not yet identified site on HBP is involved in the binding of lipid A/LPS. Proteins 2001;42:442–451.

Crystallization and molecular replacement solution of human heparin binding protein

The highly glycosylated protein, human heparin binding protein, has been crystallized in the primitive orthorhombic space group P2(1)2(1)2(1) with cell dimensions a = 39.0, b = 66.2 and c = 101.4 A. Ethanol was used as precipitant and glycerol as additive. A full data set has been collected to 3.1 A and diffraction was observed to at least 2.3 A. A molecular replacement solution using human neutrophile elastase as a search model was obtained, showing one molecule per asymmetric unit. The crystal packing showed no bad contacts and the R factor was 44.8% after ten cycles of rigid-body refinement.