Expression, purification and initial characterization of human meprin ß from Pichia pastoris.

Human meprin ß (h-meprin ß), a single-zinc metalloendoprotease of the astacin family, is potentially involved in disorders such as fibrosis and Alzheimer's disease. Here, we describe the expression of the enzyme in the yeast Pichia pastoris. The N-terminal signal sequence was replaced by the α-leader of Saccharomyces, enabling efficient secretion of the mature enzyme, harboring either an N-terminal or C-terminal His-tag. The purification by affinity and hydrophobic interaction chromatography resulted in isolation of 58.4 mg/l of homogenous human pro-meprin ß from fermentation broth. The activated enzyme isolated from yeast (yh-meprin ß) displayed virtually identical enzymatic activity as h-meprin from a mammalian cell line. Furthermore, the yh-meprin ß was N-glycosylated and secreted as a dimer with a molecular mass of 148 kDa. Endoglycosidase H treatment generated a protein with a molecular mass of 133 kDa, but essentially unchanged kinetic parameters. Thus, our data suggest that human meprin ß expressed in P. pastoris displays virtually identical parameters as meprin from other sources. The high yield of protein expression, the ease of purification and the deglycosylation in its native state appear to favor further studies aiming at inhibitor screening and structure-based inhibitor refinement.

Investigation of glucose-dependent insulinotropic polypeptide-(1-42) and glucagon-like peptide-1-(7-36) degradation in vitro by dipeptidyl peptidase IV using matrix-assisted laser desorption/ionization-time of flight mass spectrometry. A novel kinetic approach.

The incretins glucose-dependent insulinotropic polypeptide (GIP1-42) and glucagon-like peptide-1-(7-36)-amide (GLP-17-36), hormones that potentiate glucose-induced insulin secretion from the endocrine pancreas, are substrates of the circulating exopeptidase dipeptidyl peptidase IV and are rendered biologically inactive upon cleavage of their N-terminal dipeptides. This study was designed to determine if matrix-assisted laser desorption/ionization-time of flight mass spectrometry is a useful analytical tool to study the hydrolysis of these hormones by dipeptidyl peptidase IV, including kinetic analysis. Spectra indicated that serum-incubated peptides were cleaved by this enzyme with only minor secondary degradation due to other serum protease activity. Quantification of the mass spectrometric signals allowed kinetic constants for both porcine kidney- and human serum dipeptidyl peptidase IV-catalyzed incretin hydrolysis to be calculated. The binding constants (Km) of these incretins to purified porcine kidney-derived enzyme were 1.8 +/- 0.3 and 3.8 +/- 0.3 microM, whereas the binding constants observed in human serum were 39 +/- 29 and 13 +/- 9 microM for glucose-dependent-insulinotropic polypeptide and glucagon-like peptide-1-(7-36)-amide respectively. The large range of Km values found in human serum suggests a heterogeneous pool of enzyme. The close correlation between the reported kinetic constants and those previously described validates this novel approach to kinetic analysis.

[Plasma GM-CSF concentrations in rheumatoid arthritis, systemic lupus erythematosus and spondyloarthropathy]. / GM-CSF-Plasmakonzentrationen bei rheumatoider Arthritis, systemischem Lupus erythematodes und Spondylarthropathie.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a multipotent hematopoietic growth factor, which is mainly produced by T-cells and stromal cells. Beside the stimulating effects on mature granulocytes, it induces the expression of HLA class II-antigen on synovial tissue-cells in patients with rheumatoid arthritis. The concentrations of GM-CSF in the plasma of 87 patients with rheumatoid arthritis, 48 patients with spondyloarthropathy, 17 patients with systemic lupus erythematosus (SLE), and 43 healthy control persons were investigated. We used an immunoradiometric assay (IR-MA) with a detection limit of 30 pg/ml to measure the GM-CSF concentrations in plasma. The GM-CSF levels of 29 patients with severe rheumatoid arthritis (366 +/- 61 pg/ml, p less than 0.05), 58 patients with moderate rheumatoid arthritis (376 +/- 44 pg/ml, p less than 0.0001), and of 17 patients with SLE (256 +/- 41 pg/ml, p less than 0.05) were elevated compared to the control group (174 +/- 18 pg/ml). No significant differences in the mean GM-CSF plasma levels between the patients with spondyloarthropathy (190 +/- 32 pg/ml) and the control group were found. GM-CSF concentrations as high as 1300 pg/ml were detected in the synovial fluids of patients with rheumatoid arthritis. GM-CSF concentrations in the plasma of patients with severe rheumatoid arthritis were correlated with the plasma concentrations of the soluble interleukin-2-receptor (sCD25) (R = +0.53).

Is soluble CD25 antigen (interleukin-2 receptor) a useful parameter for differential diagnosis of thyrotoxicosis?

Soluble CD25 antigen was measured in 28 patients with Graves' disease and 20 patients with thyroid autonomy in order to address the question of whether this parameter could be used in the differential diagnosis of thyrotoxicosis. Soluble CD25 was significantly elevated in active Graves' disease (2430 +/- 442 U/ml, mean +/- SEM) compared to patients with thyroid autonomy (1295 +/- 225 U/ml, mean +/- SEM). However, compared to normal controls (mean 605 +/- 49 U/ml), both groups of patients had significantly elevated CD25 plasma levels. Investigations in thyroidectomized thyroid cancer patients on and off T4 suppressive therapy showed no influence of T4 on the CD25 level. Soluble CD25 concentrations did not differ in thyroid cancer patients compared to normal controls. We conclude that soluble CD25 may indicate a stimulation of the immune system with high sensitivity; however, due to the low specificity of elevated CD25 levels, its usefulness for differential diagnosis of thyrotoxicosis is limited.

Elevated TNF receptor plasma concentrations in patients with rheumatoid arthritis.

Two types of tumor necrosis factor receptors have been characterized, both capable of transmitting the signal and exerting the biological functions of TNF and lymphotoxin. We measured the plasma concentrations of two types of TNF binding proteins (sTNFR-A and sTNFR-B) in patients with rheumatoid arthritis (RA) and spondylarthropathies (SpA) using an enzyme-linked binding assay. In normal controls (n = 43), mean plasma concentrations were 1030 +/- 55 and 1461 +/- 59 pg/ml for sTNFR types A and B, respectively. In 67 patients with moderate RA, mean levels were 1422 +/- 82 pg/ml (type A) and 2088 +/- 109 pg/ml (type B); in 34 patients with severe RA, 2588 +/- 279 pg/ml and 4494 +/- 550 pg/ml, respectively, were measured (P less than 0.0001 compared to normal controls). Concentrations of both type A and type B sTNFR were highly correlated in severe RA (R2 = 0.7) but not in SpA or normal controls. T lymphocytes in synovial fluid of patients with RA expressed predominantly type A TNF receptors on their surface; in some patients a weaker expression of type B receptors was also detectable. Soluble TNF binding proteins in patients with RA were able to neutralize TNF in a cytotoxicity assay, demonstrating their ability to act as "TNF-inhibiting factors". We conclude that both types of TNF receptors are parameters of disease activity in RA and may also act as TNF antagonists.