alpha-chymotrypsin and Proteinuria

Trypsin is a protease, which is commonly used for the digestion of protein samples in proteomic experiments. The process of trypsin autolysis is known to produce autolytic peptides as well as active enzyme forms with one or more intra-chain splits. In consequence, their variable presence can influence the digestion of a protein substrate in the reaction mixture. Besides two major and well-studied forms named β-trypsin and α-trypsin, there are also other active trypsin forms known such as γ-trypsin and pseudotrypsin (ψ-trypsin). In this work, the cleavage specificity of ψ-trypsin was evaluated using in-gel digestion of protein standards followed by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) and tandem mass spectrometry (MS/MS) analyses of the resulting peptides. The numbers of produced and matching peptides were similar to those obtained using α-/β-trypsin. The same experience was obtained with a real complex protein sample from rat urine. In previous reports, ψ-trypsin was supposed to generate non-specific cleavages, which has now been reevaluated. Purified ψ-trypsin cleaved all analyzed proteins preferentially on the C-terminal side of Lys and Arg residues in accordance with the canonical tryptic cleavage. However, a minor nonspecific cleavage performance was also registered (particularly after Tyr and Phe), which was considerably higher than in the case of trypsin itself.

Topics: Animals; Autolysis; Chymotrypsin; Proteins; Proteinuria; Rats; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Trypsin

In order to present an antigen to T-cells, the antigen must first be degraded by proteasomes. Following exposure to interferons, some proteasome subunits (ss1,ss2,ss5) are replaced by others (LMP2, LMP7, MECL-1) that have more optimal catalytic properties for peptide presentation; this more efficient organelle is termed the immuno-proteasome. Here we measured gene expression of various subunits in peripheral mononuclear cells of patients with IgA nephropathy, a disease with features of immune dysregulation. We used quantitative PCR to measure the expression of proteasomal subunit mRNA in mononuclear cells from IgA nephropathy patients, a group of proteinuric control patients with idiopathic nephrotic syndromes, and healthy controls. A significant switch in the expression of trypsin- and chymotrypsin-like proteasome subunits to corresponding immuno-proteasome subunits was found in patients as compared to healthy controls. Further, we found that nuclear translocation of NF-kappaB p50 and p65 was significantly greater in the IgA nephropathy patients, but this did not correlate with the switch to the immuno-proteasome phenotype. Patients with proteinuria greater than 0.5 g/1.73 m(2)/day had a significant switch of the chymotryptic-like beta5 protease to the LMP7 subunit, but this did not occur in patients with idiopathic nephrotic syndrome. The switch to an immuno-proteasome in peripheral blood mononuclear cells of patients with IgA nephropathy suggests an increased efficiency of antigen processing and presentation. This switch appears to be independent of a coincidental activation of the NF-kappaB pathway but is associated with high levels of proteinuria, a well known risk factor for progression of IgA nephropathy.

Topics: Active Transport, Cell Nucleus; Adolescent; Adult; Antigen Presentation; Blood Cells; Case-Control Studies; Child; Child, Preschool; Chymotrypsin; Female; Glomerulonephritis, IGA; Humans; Leukocytes, Mononuclear; Male; Multienzyme Complexes; NF-kappa B; Proteasome Endopeptidase Complex; Protein Subunits; Proteinuria; RNA, Messenger; Up-Regulation; Young Adult

Topics: Amides; Amino Acid Sequence; Amino Acids; Biological Evolution; Cadmium Poisoning; Carboxypeptidases; Chromatography, Gel; Chromatography, Ion Exchange; Chromatography, Thin Layer; Chymotrypsin; Dansyl Compounds; Dithiothreitol; Electrophoresis, Paper; Globulins; Humans; Immunoglobulin G; Iodoacetates; Pepsin A; Peptides; Proteinuria; Subtilisins; Thermolysin; Thiocyanates; Trypsin

Analysis of the primary structure of beta(2)-microglobulin indicates that this human protein is homologous in sequence to the constant portion of immunoglobulin light chains (C(L)), and to the homology regions (C(H)1, C(H)2, and C(H)3) of the constant portion of gamma1 (heavy) chains of immunoglobulin G. Homology with the C(H)3 region is particularly striking. No convincing homology could be demonstrated by similar comparisons with the variable regions of immunoglobulin light and heavy chains. beta(2)-Microglobulin contains an intrachain disulfide loop of 57 amino-acid residues that is similar in size to disulfide loops found in the constant regions of immunoglobulin G. These findings suggest that beta(2)-microglobulin is a free immunoglobulin domain, possibly serving an effector function similar to that of the C(H)3 domain of gamma1 chains of immunoglobulin G.

Topics: Amino Acid Sequence; Animals; Blood Proteins; Cadmium Poisoning; Chymotrypsin; Humans; Immunoglobulin G; Immunoglobulins; Iodine Isotopes; Leukocytes; Pepsin A; Poisoning; Protein Binding; Proteinuria; Rabbits; Thermolysin; Trypsin

Topics: Acrylamides; Amino Acids; Animals; Carboxypeptidases; Chromatography, Gel; Chromatography, Ion Exchange; Chymotrypsin; Humans; Kinetics; L-Lactate Dehydrogenase; Muscles; Peptides; Proteinuria; Rabbits; Spectrophotometry; Spectrophotometry, Ultraviolet; Subtilisins; Trypsin

Topics: Chromatography, Gel; Chymotrypsin; gamma-Globulins; Humans; Molecular Weight; Myoglobin; Ovalbumin; Proteinuria; Serum Albumin; Ultracentrifugation; Urine