Roles of the P1, P2, and P3 residues in determining inhibitory specificity of kallistatin toward human tissue kallikrein

Kallistatin is a serpin with a unique P1 Phe, which confers an excellent inhibitory specificity toward tissue kallikrein. In this study, we investigated the P3-P2-P1 residues (residues 386-388) of human kallistatin in determining inhibitory specificity toward human tissue kallikrein by site-directed mutagenesis and molecular modeling. Human kallistatin mutants with 19 different amino acid substitutions at each P1, P2, or P3 residue were created and purified to compare their kallikrein binding activity. Complex formation assay showed that P1 Arg, P1 Phe (wild type), P1 Lys, P1 Tyr, P1 Met, and P1 Leu display significant binding activity with tissue kallikrein among the P1 variants. Kinetic analysis showed the inhibitory activities of the P1 mutants toward tissue kallikrein in the order of P1 Arg > P1 Phe > P1 Lys >/= P1 Tyr > P1 Leu >/= P1 Met. P1 Phe displays a better selectivity for human tissue kallikrein than P1 Arg, since P1 Arg also inhibits several other serine proteinases. Heparin distinguishes the inhibitory specificity of kallistatin toward kallikrein versus chymotrypsin. For the P2 and P3 variants, the mutants with hydrophobic and bulky amino acids at P2 and basic amino acids at P3 display better binding activity with tissue kallikrein. The inhibitory activities of these mutants toward tissue kallikrein are in the order of P2 Phe (wild type) > P2 Leu > P2 Trp > P2 Met and P3 Arg > P3 Lys (wild type). Molecular modeling of the reactive center loop of kallistatin bound to the reactive crevice of tissue kallikrein indicated that the P2 residue required a long and bulky hydrophobic side chain to reach and fill the hydrophobic S2 cleft generated by Tyr(99) and Trp(219) of tissue kallikrein. Basic amino acids at P3 could stabilize complex formation by forming electrostatic interaction with Asp(98J) and hydrogen bond with Gln(174) of tissue kallikrein. Our results indicate that tissue kallikrein is a specific target proteinase for kallistatin.     

A positively charged loop on the surface of kallistatin functions to enhance tissue kallikrein inhibition by acting as a secondary binding site for kallikrein

Kallistatin is a serine proteinase inhibitor (serpin) that specifically inhibits tissue kallikrein. The inhibitory activity of kallistatin is abolished upon heparin binding. The loop between the H helix and C2 sheet of kallistatin containing clusters of basic amino acid residues has been identified as a heparin-binding site. In this study, we investigated the role of the basic residues in this region in tissue kallikrein inhibition. Kallistatin mutants containing double Ala substitutions for these basic residues displayed a 70-80% reduction of association rate constants, indicating the importance of these basic residues in tissue kallikrein inhibition. A synthetic peptide derived from the sequence between the H helix and C2 sheet of kallistatin was shown to suppress the kallistatin-kallikrein interaction through competition for tissue kallikrein binding. To further evaluate the function of this loop, we used alpha1-antitrypsin, a non-heparin-binding serpin and slow tissue kallikrein inhibitor as a scaffold to engineer kallikrein inhibitors. An alpha1-antitrypsin chimera harboring the P3-P2' residues and a sequence homologous to the positively charged region between the H helix and C2 sheet of kallistatin acquired heparin-suppressed inhibitory activity toward tissue kallikrein and exhibited an inhibitory activity 20-fold higher than that of the other chimera, which contained only kallistatin's P3-P2' sequence, and 2300-fold higher than that of wild-type alpha1-antitrypsin. The alpha1-antitrypsin chimera with inhibitory characteristics similar to those of kallistatin demonstrates that the loop between the H helix and C2 sheet of kallistatin is crucial in tissue kallikrein inhibition, and this functional loop can be used as a module to enhance the inhibitory activity of a serpin toward tissue kallikrein. In conclusion, our results indicate that a positively charged loop between the H helix and C2 sheet of a serpin can accelerate the association of a serpin with tissue kallikrein by acting as a secondary binding site.     

Identification of a major heparin-binding site in kallistatin

Kallistatin is a heparin-binding serine proteinase inhibitor (serpin), which specifically inhibits human tissue kallikrein by forming a covalent complex. The inhibitory activity of kallistatin is blocked upon its binding to heparin. In this study we attempted to locate the heparin-binding site of kallistatin using synthetic peptides derived from its surface regions and by site-directed mutagenesis of basic residues in these surface regions. Two synthetic peptides, containing clusters of positive-charged residues, one derived from the F helix and the other from the region encompassing the H helix and C2 sheet of kallistatin, were used to assess their heparin binding activity. Competition assay analysis showed that the peptide derived from the H helix and C2 sheet displayed higher and specific heparin binding activity. The basic residues in both regions were substituted to generate three kallistatin double mutants K187A/K188A (mutations in the F helix) and K307A/R308A and K312A/K313A (mutations in the region between the H helix and C2 sheet), using a kallistatin P1Arg variant as a scaffold. Analysis of these mutants by heparin-affinity chromatography showed that the heparin binding capacity of the variant K187A/K188A was not altered, whereas the binding capacity of K307A/R308A and K312A/K313A mutants was markedly reduced. Titration analysis with heparin showed that the K312A/K313A mutant has the highest dissociation constant. Like kallistatin, the binding activity of K187A/K188A to tissue kallikrein was blocked by heparin, whereas K307A/R308A and K312A/K313A retained significant binding and inhibitory activities in the presence of heparin. These results indicate that the basic residues, particularly Lys(312)-Lys(313), in the region between the H helix and C2 sheet of kallistatin, comprise a major heparin-binding site responsible for its heparin-suppressed tissue kallikrein binding.     

Kallistatin: a novel human tissue kallikrein inhibitor. Purification, characterization, and reactive center sequence.

A novel human tissue kallikrein inhibitor designated as kallistatin has been purified from plasma to apparent homogeneity by polyethylene glycol fractionation and successive chromatography on heparin-Agarose, DEAE-Sepharose, hydroxylapatite, and phenyl-Superose columns. A purification factor of 4350 was achieved with a yield of approximately 1.35 mg per liter of plasma. The purified inhibitor migrates as a single band with an apparent molecular mass of 58 kDa when analyzed on SDS-polyacrylamide gel electrophoresis under reducing conditions. It is an acidic protein with pI values ranging from 4.6 to 5.2. No immunological cross-reactivity was found by Western blot analyses between kallistatin and other serpins. Kallistatin inhibits human tissue kallikrein's activity toward kininogen and tripeptide substrates. The second-order reaction rate constant (ka) was determined to be 2.6 x 10(4) M-1 s-1 using Pro-Phe-Arg-MCA. The inhibition is accompanied by formation of an equimolar, heat- and SDS-stable complex between tissue kallikrein and kallistatin, and by generation of a small carboxyl-terminal fragment from the inhibitor due to cleavage at the reactive site by tissue kallikrein. Heparin blocks kallistatin's complex formation with tissue kallikrein and abolishes its inhibitory effect on tissue kallikrein's activity. The amino-terminal residue of kallistatin is blocked. Sequence analysis of the carboxyl-terminal fragment generated from kallistatin reveals the reactive center sequence from P1' to P15', which shares sequence similarity with, but is different from known serpins including protein C inhibitor, alpha 1-antitrypsin, and alpha 1-antichymotrypsin. The results show that kallistatin is a new member of the serpin superfamily that inhibits human tissue kallikrein.     

Interaction of subtilisin BPN' and recombinant fungal protease inhibitor F from silkworm with substituted P1 site residues

Fungal protease inhibitor F (FPI-F) from silkworm inhibits subtilisin and fungal proteases. FPI-F mutants P(1) residues of which, Thr(29), were replaced with Glu, Phe, Gly, Leu, Met, and Arg, were prepared. The inhibitory activities of mutated FPI-F against subtilisin and other mammalian proteases indicated that FPI-F might be a specific inhibitor toward subtilisin-type protease.     

Inhibition of subtilisin BPN' by reaction site P1 mutants of Streptomyces subtilisin inhibitor

It has been shown that the P1 site (the center of the reactive site) of protease inhibitors corresponds to the specificity of the cognate protease, and consequently specificity of Streptomyces subtilisin inhibitor (SSI) can be altered by substitution of a single amino acid at the P1 site. In this paper, to investigate whether similar correlation between inhibitory activity of mutated SSI and substrate preference of protease is observed for subtilisin BPN', which has broad substrate specificity, a complete set of mutants of SSI at the reaction site P1 (position 73) was constructed by cassette and site-directed mutagenesis and their inhibitory activities toward subtilisin BPN' were measured. Mutated SSIs which have a polar (Ser, Thr, Gln, Asn), basic (Lys, Arg), or aromatic amino acid (Tyr, Phe, Trp, His), or Ala or Leu, at the P1 site showed almost the same strong inhibitory activity toward subtilisin as the wild type (Met) SSI. However, the inhibitory activity of SSI variants with an acidic (Glu, Asp), or a beta-branched aliphatic amino acid (Val, Ile), or Gly or Pro, at P1 was decreased. The values of the inhibitor constant (Ki) of mutated SSIs toward subtilisin BPN' were consistent with the substrate preference of subtilisin BPN'. A linear correlation was observed between log(1/Ki) of mutated SSIs and log(1/Km) of synthetic substrates. These results demonstrate that the inhibitory activities of P1 site mutants of SSI are linearly related to the substrate preference of subtilisin BPN', and indicate that the binding mode of the inhibitors with the protease may be similar to that of substrates, as in the case of trypsin and chymotrypsin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Site-directed mutageneses of rat liver cytochrome P-450d: catalytic activities toward benzphetamine and 7-ethoxycoumarin

Catalytic activities toward benzphetamine and 7-ethoxycoumarin of 11 distal mutants, 9 proximal mutants, and 3 aromatic mutants of rat liver cytochrome P-450d were studied. A distal mutant Thr319Ala was not catalytically active toward benzphetamine, while this mutant retained activity toward 7-ethoxycoumarin. Distal mutants Gly316Glu, Thr319Ala, and Thr322Ala displayed higher activities (kcat/Km) toward 7-ethoxycoumarin that were 2.4-4.7-fold higher than that of the wild-type enzyme. Although kcat/Km values of four multiple distal mutants toward benzphetamine were less than half that of the wild type, activities of these mutants toward 7-ethoxycoumarin were almost the same as or higher than the wild-type activity toward this substrate. The distal double mutant Glu318Asp, Phe325Tyr showed 6-fold higher activity than the wild-type P-450d toward 7-ethoxycoumarin. Activities of the proximal mutants Lys453Glu and Arg455Gly toward both substrates were much lower (less than one-seventh) than the corresponding wild-type activities. Catalytic activities of three aromatic mutants, Phe425Leu, Pro427Leu, and Phe430Leu, toward benzphetamine were less than 7% of that of the wild type, while the activities of these aromatic mutants toward 7-ethoxycoumarin were more than 2.5 times higher than the wild-type activity toward this substrate. From these findings, in conjunction with a molecular model for P-450d, we suggest that (1) the relative importance to catalysis of various distal helix amino acids differs depending on the substrate and that these differences are associated with the size, shape, and flexibility of the substrate and (2) the proximal residue Lys453 appears to play a critical role in the catalytic activity of P-450d, perhaps by participating in forming an intermolecular electron-transfer complex.     

Interaction of Subtilisin BPN′ and Recombinant Fungal Protease Inhibitor F from Silkworm with Substituted P1 Site Residues

Fungal protease inhibitor F (FPI-F) from silkworm inhibits subtilisin and fungal proteases. FPI-F mutants P₁ residues of which, Thr²⁹, were replaced with Glu, Phe, Gly, Leu, Met, and Arg, were prepared. The inhibitory activities of mutated FPI-F against subtilisin and other mammalian proteases indicated that FPI-F might be a specific inhibitor toward subtilisin-type protease.     

Structural elements of kallistatin required for inhibition of angiogenesis

Kallistatin is aserpin first identified as a specific inhibitor of tissue kallikrein.Our recent studies showed that kallikrein promoted angiogenesis,whereas kallistatin inhibited angiogenesis and tumor growth. This studyis aimed to identify the structural elements of kallistatin essentialfor its antiangiogenic function. Kallistatin mutants at the hingeregion (A377T) and a major heparin-binding domain (K312A/K313A) werecreated by site-directed mutagenesis. Recombinant kallistatin mutantA377T did not bind or inhibit tissue kallikrein activity. Wild-typekallistatin and kallistatin mutant A377T, but not kallistatin mutantK312A/K313A lacking heparin-binding activity, inhibited VEGF-inducedproliferation, growth, and migration of human microvascular endothelialcells. Similarly, wild-type kallistatin and kallistatin mutant A337T,but not kallistatin mutant K312A/K313A, significantly inhibitedVEGF-induced capillary tube formation of cultured endothelial cells inMatrigel and capillary formation in Matrigel implants in mice. Toelucidate the role of the heparin-binding domain in modulatingangiogenesis, we showed that wild-type kallistatin interrupted thebinding of ¹²⁵I-labeled VEGF to endothelial cells, whereaskallistatin mutant K312A/K313A did not interfere with VEGF binding.Consequently, wild-type kallistatin, but not kallistatin mutantK312A/K313A, suppressed VEGF-induced phosphorylation of Akt. Takentogether, these results indicate that the heparin-binding domain, butnot the reactive site loop of kallistatin, is essential for inhibiting VEGF-induced angiogenesis.

     

Functional role of putative critical residues in Mycobacterium tuberculosis RNase P protein

RNase P is involved in processing the 5⿲ end of pre-tRNA molecules. Bacterial RNase P contains a catalytic RNA subunit and a protein subunit. In this study, we have analyzed the residues in RNase P protein of M. tuberculosis that differ from the residues generally conserved in other bacterial RNase Ps. The residues investigated in the current study include the unique residues, Val27, Ala70, Arg72, Ala77, and Asp124, and also Phe23 and Arg93 which have been found to be important in the function of RNase P protein components of other bacteria. The selected residues were individually mutated either to those present in other bacterial RNase P protein components at respective positions or in some cases to alanine. The wild type and mutant M. tuberculosis RNase P proteins were expressed in E. coli, purified, used to reconstitute holoenzymes with wild type RNA component in vitro, and functionally characterized. The Phe23Ala and Arg93Ala mutants showed very poor catalytic activity when reconstituted with the RNA component. The catalytic activity of holoenzyme with Val27Phe, Ala70Lys, Arg72Leu and Arg72Ala was also significantly reduced, whereas with Ala77Phe and Asp124Ser the activity of holoenzyme was similar to that with the wild type protein. Although the mutants did not suffer from any binding defects, Val27Phe, Ala70Lys, Arg72Ala and Asp124Ser were less tolerant towards higher temperatures as compared to the wild type protein. The K_m of Val27Phe, Ala70Lys, Arg72Ala and Ala77Phe were >2-fold higher than that of the wild type, indicating the substituted residues to be involved in substrate interaction. The study demonstrates that residues Phe23, Val27 and Ala70 are involved in substrate interaction, while Arg72 and Arg93 interact with other residues within the protein to provide it a functional conformation.     

Chemical replacement of P1' arginine residue at the first reactive site of peanut protease inhibitor B-III

The contribution of the P1' residue at the first reactive site of peanut protease inhibitor B-III to the inhibition was analyzed by replacement of the P1' Arg(11) with other amino acids (Arg, Ser, Ala, Leu, Phe, Asp) after selective modification of the second reactive site. The Arg derivative had the same trypsin inhibitory activity as the native inhibitor (Ki = 2 X 10(-9) M). The Ser derivative inhibited more weakly (Ki = 2 X 10(-8) M). The Ala and Leu derivatives inhibited trypsin very weakly (Ki = 2 X 10(-7) M and 4 X 10(-7) M, respectively), and the Phe and Asp derivatives not at all. These results suggest that the P1' arginine residue is best for inhibitory activity at the first reactive site of B-III, although it has been suggested that a P1' serine residue at the reactive site is best for inhibitory activity of Bowman-Birk type inhibitors.     

Engineered NADH-dependent GRE2 from Saccharomyces cerevisiae by directed enzyme evolution enhances HMF reduction using additional cofactor NADPH

Furfural and 5-hydroxymethylfurfural (HMF) are inhibitors generated by lignocellulosic biomass pretreatment such as dilute acid hydrolysis that inhibit microbial growth and interfere with subsequent fermentation. It is possible to in situ detoxify these inhibitory compounds by aldehyde reductions using tolerant Saccharomyces cerevisiae. YOL151W (GRE2) is a commonly recognized up-regulated gene expressed under stress conditions that encodes reductase activities toward furfural and HMF using cofactor NADH. Applying a directed enzyme evolution approach, we altered the genetic code of GRE2 yielding two mutants with amino acid substitutions of Gln261 to Arg261 and Phe283 to Leu283; and Ile107 to Val107, Gln261 to Arg261, and Val285 to Asp285 for strain Y62-C11 and Y62-G6, respectively. Clones of these mutants showed faster growth rates and were able to establish viable cultures under 30 mM HMF challenges when compared with a wild type GRE2 clone when inoculated into synthetic medium containing this inhibitor. Compared with the wild type control, crude cell extracts of the two mutants showed 3- to 4-fold and 3- to 9-fold increased specific enzyme activity using NADH toward HMF and furfural reduction, respectively. While retaining its aldehyde reductase activities using the cofactor NADH, mutant Y62-G6 displayed significantly greater reductase activities using NADPH as the cofactor with 13- and 15-fold increase toward furfural and HMF, respectively, as measured by its partially purified protein. Using reverse engineering and site directed mutagenesis methods, we were able to confirm that the amino acid substitution of the Asp285 is responsible for the increased aldehyde reductase activities by utilizing the additional cofactor NADPH.     

Roles of the N-terminal domain and remote substrate binding subsites in activity of the debranching barley limit dextrinase

Barley limit dextrinase (HvLD) of glycoside hydrolase family 13 is the sole enzyme hydrolysing α-1,6-glucosidic linkages from starch in the germinating seed. Surprisingly, HvLD shows 150- and 7-fold higher activity towards pullulan and β-limit dextrin, respectively, than amylopectin. This is investigated by mutational analysis of residues in the N-terminal CBM-21-like domain (Ser14Arg, His108Arg, Ser14Arg/His108Arg) and at the outer subsites +2 (Phe553Gly) and +3 (Phe620Ala, Asp621Ala, Phe620Ala/Asp621Ala) of the active site. The Ser14 and His108 mutants mimic natural LD variants from sorghum and rice with elevated enzymatic activity. Although situated about 40 Å from the active site, the single mutants had 15–40% catalytic efficiency compared to wild type for the three polysaccharides and the double mutant retained 27% activity for β-limit dextrin and 64% for pullulan and amylopectin. These three mutants hydrolysed 4,6-O-benzylidene-4-nitrophenyl-6³-α-d-maltotriosyl-maltotriose (BPNPG3G3) with 51–109% of wild-type activity. The results highlight that the N-terminal CBM21-like domain plays a role in activity. Phe553 and the highly conserved Trp512 sandwich a substrate main chain glucosyl residue at subsite +2 of the active site, while substrate contacts of Phe620 and Asp621 at subsite +3 are less prominent. Phe553Gly showed 47% and 25% activity on pullulan and BPNPG3G3, respectively having a main role at subsite +2. By contrast at subsite +3, Asp621Ala increased activity on pullulan by 2.4-fold, while Phe620Ala/Asp621Ala retained only 7% activity on pullulan albeit showed 25% activity towards BPNPG3G3. This outcome supports that the outer substrate binding area harbours preference determinants for the branched substrates amylopectin and β-limit dextrin.     

Structure-function analysis of the prosegment of the proprotein convertase PC5A

To investigate if some residues within the prosegment of PC5A are important for its optimal proteolytic function, various PC5A mutants were cellularly expressed, and their processing activities were compared using pro-vascular endothelial growth factor C (pro-VEGF-C) as a substrate. Although wild type PC5A almost completely processes pro-VEGF-C, a prosegment deletion as well as both P1 mutants of the primary (R116A) and secondary (R84A) autocatalytic cleavage sites are inactive. The in vitro inhibitory potency of various decapeptides mimicking the C-terminal sequence of PC5 prosegment (pPC5) revealed that the native (107)QQVVKKRTKR(116) peptide is a nanomolar inhibitor, whereas its P6 mutant K111H is more selective toward PC5A than Furin. In vitro activity assays using the bacterially expressed pPC5 and its mutants revealed them to be very potent nanomolar inhibitors (IC(50)) and only approximately 6-fold more selective inhibitors of PC5A versus Furin. Expression of the preprosegment of PC5 (ppPC5) and its mutants in Chinese hamster ovary FD11 cells overexpressing pro-VEGF-C with either PC5A or Furin showed them to be as good inhibitors of PC5A as the serpin alpha1-antitrypsin Portland (alpha1-PDX), ppFurin, or ppPACE4 but less potent toward overexpressed Furin. In conclusion, cleavages of the prosegment of PC5A at both Arg(116) and Arg(84) are required for PC5A cellular activity, and ppPC5 is a very potent but modestly selective cellular inhibitor of PC5A.     

Novel roles of kallistatin, a specific tissue kallikrein inhibitor, in vascular remodeling

We have purified, cloned and characterized kallistatin, a tissue kallikrein-binding protein (KBP) in humans and rodents. Kallistatin is a unique serine proteinase inhibitor (serpin) with Phe-Phe residues at the P2 and P1 positions. Structural and functional analysis of kallistatin by site-directed mutagenesis and protein engineering indicate that wild-type kallistatin is selective for tissue kallikrein. Kallistatin is expressed and localized in endothelial and smooth muscle cells of blood vessels and has multiple roles in vascular function independent of the tissue kallikrein-kinin system. First, kallistatin induces vasorelaxation of isolated aortic rings and reduces renal perfusion pressure in isolated rat kidneys. Transgenic mice overexpressing rat kallistatin are hypotensive, and adenovirus-mediated gene delivery of human kallistatin attenuates blood pressure rise in spontaneously hypertensive rats. Second, kallistatin stimulates the proliferation and migration of vascular smooth muscle cells in vitro and neointima formation in balloon-injured rat arteries. Third, kallistatin inhibits the proliferation, migration and adhesion of endothelial cells in vitro and angiogenesis in the rat model of hindlimb ischemia. These results demonstrate novel roles of kallistatin in blood pressure regulation and vascular remodeling.     

Chromogenic substrates of bovine beta-trypsin: the influence of an amino acid residue in P1 position on their interaction with the enzyme

The Cucurbita maxima trypsin inhibitor CMTI-III molecule was used as a vehicle to design and synthesize a series of trypsin chromogenic substrates modified in position P1: Ac-Ala-Val-Abu-Pro-X-pNA, where X = Orn, Lys, Arg, Har, Arg(NO(2)), Cit, Hci, Phe(p-CN), Phe(p-NH(2)); pNA = p-nitroanilide. The most active compounds (as determined by specificity constant k(cat)/K(m)) were peptides with the Arg and Lys residues in the position discussed. Changes in the length and the decrease of the positive charge of the amino acid residue side chain in position P(1) resulted in the decrease or loss of the affinity towards bovine beta-trypsin. Among peptides containing amino acid residues with uncharged side chains in position P1, only one with p-cyano-l-Phe revealed activity. These results correspond well with trypsin inhibitory activity of CMTI-III analogues modified in the equivalent position, indicating the same type of interaction between position P1 of the substrate or inhibitor and S1 site specificity of trypsin.     

Characterization of modeled inhibitory binding sites on isoform one of the Na+/H+ exchanger

Mammalian Na⁺/H⁺ exchanger isoform one (NHE1) is a plasma membrane protein responsible for pH regulation in mammalian cells. Excess activity of the protein promotes heart disease and is a trigger of metastasis in cancer. Inhibitors of the protein exist but problems in specificity have delayed their clinical application. Here we examined amino acids involved in two modeled inhibitor binding sites (A, B) in human NHE1. Twelve mutations (Asp159, Phe348, Ser351, Tyr381, Phe413, Leu465, Gly466, Tyr467, Leu468, His473, Met476, Leu481) were made and characterized. Mutants S351A, F413A, Y467A, L468A, M476A and L481A had 40–70% of wild type expression levels, while G466A and H473A expressed 22% ~ 30% of the wild type levels. Most mutants, were targeted to the cell surface at levels similar to wild type NHE1, approximately 50–70%, except for F413A and G466A, which had very low surface targeting. Most of the mutants had measurable activity except for D159A, F413A and G466A. Resistance to inhibition by EMD87580 was elevated in mutants F438A, L465A and L468A and reduced in mutants S351A, Y381A, H473A, M476A and L481A. All mutants with large alterations in inhibitory properties showed reduced Na⁺ affinity. The greatest changes in activity and inhibitor sensitivity were in mutants present in binding site B which is more closely associated with TM4 and C terminal of extracellular loop 5, and is situated between the putative scaffolding domain and transport domain. The results help define the inhibitor binding domain of the NHE1 protein and identify new amino acids involved in inhibitor binding.     

The LRRK2 Gly2385Arg variant is associated with Parkinson’s disease: genetic and functional evidence

Evidence of LRRK2 haplotypes associated with Parkinson’s disease (PD) risk was recently found in the Chinese population from Singapore, and a common LRRK2 missense variant, Gly2385Arg, was independently detected as a putative risk factor for PD in the Chinese population from Taiwan. To test the association between the Gly2385Arg variant in a large case-control sample of Chinese ethnicity from Singapore, and to perform functional studies of the wild type and Gly2385Arg LRRK2 protein in human cell lines. In a case-control study involving 989 Chinese subjects, the frequency of the heterozygous Gly2385Arg genotype was higher in PD compared to controls (7.3 vs. 3.6%, odds ratio = 2.1, 95% CI: 1.1–3.9, P = 0.014); these values yield an estimated population attributable risk (PAR) of ∼4%. In a multivariate logistic regression analysis with the disease group (PD vs. controls) as the dependent variable and the genotype as an independent factor with adjustments made for the effect of age and gender, the heterozygous Gly2385Arg genotype remained associated with an increased risk of PD compared to wild type genotype (odds ratio = 2.67, 95% CI: 1.43–4.99, P = 0.002). The glycine at position 2385 is a candidate site for N-myristoylation, and the Gly2385Arg variant replaces the hydrophobic glycine with the hydrophilic arginine, and increases the net positive charge of the LRRK2 WD40 domain. In transfection studies, we demonstrated that both the wild type and Gly2385Arg variant LRRK2 protein localize to the cytoplasm and form aggregates. However, under condition of oxidative stress, the Gly2385Arg variant was more toxic and associated with a higher rate of apoptosis. Our study lends support to the contention that the Gly2385Arg is a common risk factor for PD in the Chinese population. Our bioinformatics and in-vitro studies also suggest that the Gly2385Arg variant is biologically relevant and it might act through pro-apoptotic mechanisms.     

Recombinant aprotinin homologue with new inhibitory specificity for cathepsin G.

The substitution of amino acids in the reactive site of aprotinin, a bovine serine proteinase inhibitor with potent activity against trypsin, plasmin and tissue kallikrein, led to a change in specificity of the inhibitor. Twelve new aprotinin variants prepared by recombinant DNA technology and expressed in Escherichia coli clearly demonstrated that the neighbouring groups of the P1 residue, in particular P'2, contribute to the specificity of the inhibitor, while earlier investigations on semisynthetically prepared variants revealed the importance of the P1 residue in dominating the inhibitory specificity. Recombinant aprotinin variants which act specifically against chymotrypsin-like proteinases, were obtained by substitution of the amino acids in position P1 and P'2 by hydrophobic amino acids like phenylalanine, tyrosine and leucine. Some of these variants, particularly those with phenylalanine or leucine substitutions, were also found to exhibit inhibitory activity against cathepsin G with an equilibrium constant of dissociation Ki of 10(-8) M. Inhibitory specificity against cathepsin G was not found in any semisynthetic variant prepared earlier.