Preparative Purification of Dipeptidyl Peptidase IV

Abstract

Dipeptidyl-Peptidase IV was purified from pig kidney by ammonium sulfate fractionation, gel filtration, QAE-cellulose chromatography and affinity columns with Gly-Pro- and Concanavalin A-Sepharose. The specific activity of the purified enzyme is 41.8 units/mg. Polyacrylamide gel electrophoresis and silver staining show a single band. The enzyme preparation is free of aminopeptidase and dipeptidase activity, proved fluorimetrically and by gas chromatography/mass spectrometry. The most important procedure for removal of contaminating enzyme activities is a stepwise NaCl-gradient on a QAE-ZetaPrep ion exchange disk.     

Identification of the Gene Encoding Isoprimeverose-producing Oligoxyloglucan Hydrolase in Aspergillus oryzae

Aspergillus oryzae produces a unique β-glucosidase, isoprimeverose-producing oligoxyloglucan hydrolase (IPase), that recognizes and releases isoprimeverose (α-d-xylopyranose-(1→6)-d-glucopyranose) units from the non-reducing ends of oligoxyloglucans. A gene encoding A. oryzae IPase, termed ipeA, was identified and expressed in Pichia pastoris. With the exception of cellobiose, IpeA hydrolyzes a variety of oligoxyloglucans and is a member of the glycoside hydrolase family 3. Xylopyranosyl branching at the non-reducing ends was vital for IPase activity, and galactosylation at a α-1,6-linked xylopyranosyl side chain completely abolished IpeA activity. Hepta-oligoxyloglucan saccharide (Xyl₃Glc₄) substrate was preferred over tri- (Xyl₁Glc₂) and tetra- (Xyl₂Glc₂) oligoxyloglucan saccharides substrates. IpeA transferred isoprimeverose units to other saccharides, indicating transglycosylation activity. The ipeA gene was expressed in xylose and xyloglucan media and was strongly induced in the presence of xyloglucan endo-xyloglucanase-hydrolyzed products. This is the first study to report the identification of a gene encoding IPase in eukaryotes.     

Substance P in Human Plasma Is Degraded by Dipeptidyl Peptidase IV, Not by Cholinesterase

Human serum cleaves two dipeptides from the N-terminus of the neurohormone substance P. It has been suggested that this degrading activity is inherent to serum cholinesterase. We oppose this, because it turned out that highly purified serum cholinesterase contains traces of dipeptidyl peptidase IV, an enzyme known to attack the N-terminus of substance P. The peptidase is incompletely separated from cholinesterase during the procainamide-gel affinity chromatography as the last step of the usual purification procedure. Physostigmine completely inhibits the hydrolysis of butyrylthiocholine by such purified cholinesterase preparations, but not their substance P-degrading activity. Vice versa, epsilon-carbobenzoxy-lysylproline, an inhibitor of dipeptidyl peptidase IV, inhibits the peptidase activity of these preparations more than their esterase activity. After rechromatography on procainamide gel the peptidase is completely separated and the remaining cholinesterase has lost its substance P-degrading activity. We conclude that the N-terminal region of substance P is not degraded by cholinesterase but by the contaminating dipeptidyl peptidase IV, a different serine enzyme.     

Integrative Transformation of Aspergillus oryzae with a Plasmid Containing the Aspergillus nidulans argB Gene

The transformation of Aspergillus oryzae has been achieved with a plasmid carrying the Aspergillus nidulans argB gene coding for ornithine carbamoyltransferase (OCTase). The frequency of transformation was relatively low (0.7 transformants/μg DNA) but the transformed phenotype was extremely stable for many generations without selective pressure.

Southern blot analysis revealed that transformation had occurred by integration of multiple tandem copies of plasmid DNA into the host genome through non-homologous recombination. There was no evidence of the existence of free plasmid in the transformants. The number of integrated copies of the plasmid ranged from 15 to 60. The specific activity of OCTase in the cell- free extract was proportional to the copy number of the plasmid, indicating that most of the integrated argB gene was expressed.     

Identification of Lympho-Epithelial Kazal-Type Inhibitor 2 in Human Skin as a Kallikrein-Related Peptidase 5-Specific Protease Inhibitor

Kallikreins-related peptidases (KLKs) are serine proteases and have been implicated in the desquamation process of the skin. Their activity is tightly controlled by epidermal protease inhibitors like the lympho-epithelial Kazal-type inhibitor (LEKTI). Defects of the LEKTI-encoding gene serine protease inhibitor Kazal type (Spink)5 lead to the absence of LEKTI and result in the genodermatose Netherton syndrome, which mimics the common skin disease atopic dermatitis. Since many KLKs are expressed in human skin with KLK5 being considered as one of the most important KLKs in skin desquamation, we proposed that more inhibitors are present in human skin. Herein, we purified from human stratum corneum by HPLC techniques a new KLK5-inhibiting peptide encoded by a member of the Spink family, designated as Spink9 located on chromosome 5p33.1. This peptide is highly homologous to LEKTI and was termed LEKTI-2. Recombinant LEKTI-2 inhibited KLK5 but not KLK7, 14 or other serine proteases tested including trypsin, plasmin and thrombin. Spink9 mRNA expression was detected in human skin samples and in cultured keratinocytes. LEKTI-2 immune-expression was focally localized at the stratum granulosum and stratum corneum at palmar and plantar sites in close localization to KLK5. At sites of plantar hyperkeratosis, LEKTI-2 expression was increased. We suggest that LEKTI-2 contributes to the regulation of the desquamation process in human skin by specifically inhibiting KLK5.     

Transformation of Aspergillus aculeatus Using the Drug Resistance Gene of Aspergillus oryzae and the pyrG Gene of Aspergillus nidulans

Transformation systems for Aspergillus aculeatus has been developed, based on the use of the pyrithiamine resistance gene of Aspergillus oryzae and the orotidine-5′-monophosphate decarboxylase gene (pyrG) of Aspergillus nidulans. An A. aculeatus mutant which can be transformed effectively by the A. nidulans pyrG gene was isolated as a transformation host. This is the first report of transformation of A. aculeatus.     

Functional Expression and Characterization of Dipeptidyl Peptidase IV from the Black-Bellied Hornet Vespa basalis in Sf21 Insect Cells

The maturation of mastoparan B, the major toxin peptide in the venom of Vespa basalis, requires enzymatic cleavage of its prosequence presumably via sequential liberation of dipeptides. The putative processing enzyme, dipeptidyl peptidase IV, was expressed as a glycosylated His-tag fusion protein (rDPP-IV) via the baculovirus expression system. rDPP-IV purified by one-step nickel-affinity chromatography was verified by Western blot and LC-MS/MS analysis. The k_cat/K_m of rDPP-IV was determined to be in the range of 10–500 mM^?1·S^?1 for five synthetic substrates. The optimal temperature and pH for rDPP-IV were determined to be 50 °C and pH 9. Enzymatic activity of rDPP-IV was significantly reduced by 80 and 60% in the presence of sitagliptin and phenylmethylsulfonyl fluoride respectively.     

Purification and Properties of Acid Carboxypeptidase IV from Aspergillus oryzae

Acid carboxypeptidase IV from Aspergillus oryzae was purified from the rivanol precipitable fraction by column chromatography on DEAE-cellulose, DEAE-Sephadex A–50, hydroxylapatite and P-cellulose and gel filtration through Sephadex G–100. The optimum pH is at pH 3.0 for carbobenzoxy-l-glutamyl-l-tyrosine. The enzyme activity was inhibited by sulfhydryl reagents and diisopropylphosphorofluoridate, but was not inhibited by metal chelating agents. The molecular weight of the enzyme was estimated to be about 43,000 by gel filtration method.     

Expression of CD26 and its Association with Dipeptidyl Peptidase IV Activity in Lymphocytes of Type 2 Diabetes Patients

Immune response and inflammation were suggested to play certain roles in the development and complications of type 2 diabetes mellitus. The main objective of this study was to investigate the CD26 expression and its relationship with adenosine deaminase (ADA), dipeptidyl peptidase IV (DPP-IV), γ-glutamyltransferase (GGT), and N-acetyl-β-glucosaminidase (NAG) activities in lymphocytes of type 2 diabetics (T2DM) patients. These parameters were assessed in 25 T2DM patients and 20 control subjects. We observed a decrease in CD26 expression and a significant increase in the ADA activity in T2DM patients when compared with control subjects. There were no differences between activities of DPP-IV, NAG, and GGT in lymphocytes of T2DM patients and control subjects. Meanwhile, a significant negative correlation was observed between CD26 expression and DPP-IV activity in lymphocytes of T2DM patients. Moreover, a positive correlation was found between DPPIV and ADA activities. The results suggest that the reduction of CD26 expression may be associated in the regulation of DPP-IV in T2DM patients.     

Gene silencing by RNA interference in the koji mold Aspergillus oryzae

We found the orthologous genes required for RNA interference (RNAi) in the Aspergillus oryzae genome database, and constructed a set of tools for gene silencing using RNAi in A. oryzae. This system utilizes compatible restriction enzyme sites so that only a single target gene fragment is required to create the hairpin RNA cassette. For ease of handling, we also separated the construction of the hairpin RNA cassette for the target gene from its subsequent introduction into the expression vector. Using the brlA gene as a target for RNAi, we detected decreased mRNA levels and a delayed conidiation phenotype in the transformants. Furthermore, even though A. oryzae possesses three copies of the alpha-amylase gene, a single copy of an alpha-amylase RNAi construct was sufficient to downregulate the mRNA levels and decrease the enzymatic activity to 10% of control levels. Gene silencing by RNAi should provide a powerful genetic tool for post-genomic studies of the industrially important fungus A. oryzae.     

单宁酶基因在黑曲霉ST31中的克隆与表达

利用PCR扩增得到米曲霉(Aspergillusoryzae)单宁酶(tannase)基因的编码序列,经DNA测序证实单宁酶基因已成功克隆,然后将其连接到黑曲霉的表达载体ANED2-SP2上构建单宁酶基因表达载体。将构建好的单宁酶基因表达载体通过原生质转化法导入黑曲霉菌株ST31中进行表达研究。结果表明重组菌株的单宁酶活力最高为104.02U/ml发酵液,比原始出发菌株米曲霉提高2~3倍。研究构建了黑曲霉的高效转化体系,提高了黑曲霉表达系统的应用水平,为其它新酶的研究提供有价值的参考。     

Neutral Aminopeptidase and Dipeptidyl Peptidase IV Activities in Plasma of Monosodium Glutamate Obese and Food‐deprived Rats

Biometric parameters, glycemia and activity levels of plasma neutral aminopeptidase (APN) and dipeptidyl peptidase IV (DPPIV) were measured in monosodium glutamate obese and food‐deprived rats (MSG‐FD), to analyze the involvement of these enzymes in such situations. Plasma APN was distinguished as sensitive (PSA) (K_m = 7.8 × 10^?5 mol/l) and predominantly insensitive (APM) (K_m = 21.6 × 10^?5 mol/l) to puromycin, whereas DPPIV was sensitive (DPPIV‐DS) (K_m = 0.24 × 10^?5 mol/l) and predominantly insensitive (DPPIV‐DI) (K_m = 7.04 × 10^?5 mol/l) to diprotin A. Although unchanged in the MSG and food‐deprived animals, APM activity levels were closely correlated with body mass, Lee index, and mass of retroperitoneal fat pad in the food deprived, but not in the MSG animals. DPPIV‐DI activity levels decreased by 33% and were correlated with body mass, Lee index, and mass of periepididymal fat pad in the food‐deprived MSG rats. These data suggest that APM and DPPIV‐DI are respectively related to the downregulation of somatostatin in food‐deprived rats, and to the recovery of energy balance in MSG obese rats during food deprivation.     

High Level Secretion of Calf Chymosin Using a Glucoamylase-prochymosin Fusion Gene in Aspergillus oryzae

A recombinant chymosin was secreted at high levels using fusion genes with A. oryzae glucoamylase gene (glaA) and a wheat bran solid-state culture system. Two portions of the A. oryzae glucoamylase, one with almost the entire glucoamylase (GA1–603) lacking 9 amino acids at the carboxyl terminal, and the other (GA1–511) lacking the starch binding-domain, were fused in frame with prochymosin cDNA. Western blot analysis indicated that the mature chymosin was released from the secreted fusion protein by autocatalytic processing. The transformant harboring the GA1-511-prochymosin construct showed about 5-fold chymosin production of the transformant in which the chymosin gene was directly expressed under the control of the glaA promoter in submerged culture. Moreover, wheat bran solid-state culture gave about 500-fold higher yield of the chymosin (approximately 150 mg/kg wheat bran) compared with the submerged culture.     

Characterization of the Aspergillus nidulans nmrA Gene Involved in Nitrogen Metabolite Repression

The gene nmrA of Aspergillus nidulans has been isolated and found to be a homolog of the Neurospora crassa gene nmr-1, involved in nitrogen metabolite repression. Deletion of nmrA results in partial derepression of activities subject to nitrogen repression similar to phenotypes observed for certain mutations in the positively acting areA gene.