The stalk region of the RecU resolvase is essential for Holliday junction recognition and distortion

The Bacillus subtilis RecU protein has two activities: to recognize, distort, and cleave four-stranded recombination intermediates and to modulate RecA activities. The RecU structure shows a mushroom-like appearance, with a cap and a stalk region. The RuvB interaction and the catalytic residues are located in the cap region of dimeric RecU. We report here that the stalk region is essential not only for RecA modulation but also for Holliday junction (HJ) recognition. Two recU mutants, which map in the stalk region, were isolated and characterized. In vivo, a RecU variant with a Phe81-to-Ala substitution (F81A) was as sensitive to DNA-damaging agents as a null recU strain, and a similar substitution at tyrosine 80 (Y80A) showed an intermediate phenotype. RecUY80A and RecUF81A poorly recognize and distort HJs. RecUY80A cleaves HJs with low efficiency, and RuvB modulates cleavage. At high concentrations, RecUF81A binds to HJs but fails to cleave them. Unlike wild-type RecU, RecUY80A and RecUF81A do not inhibit RecA dATPase and strand-exchange activities. The RecU stalk region is involved in RecA interaction, but once an HJ is bound, RecU fails to modulate RecA activities. Our biochemical study provides a mechanistic basis for the connections between these two mutually exclusive stages (i.e., RecA modulation and HJ resolution) of the recombination reaction.     

The RuvA homologues from Mycoplasma genitalium and Mycoplasma pneumoniae exhibit unique functional characteristics

The DNA recombination and repair machineries of Mycoplasma genitalium and Mycoplasma pneumoniae differ considerably from those of gram-positive and gram-negative bacteria. Most notably, M. pneumoniae is unable to express a functional RecU Holliday junction (HJ) resolvase. In addition, the RuvB homologues from both M. pneumoniae and M. genitalium only exhibit DNA helicase activity but not HJ branch migration activity in vitro. To identify a putative role of the RuvA homologues of these mycoplasmas in DNA recombination, both proteins (RuvA(Mpn) and RuvA(Mge), respectively) were studied for their ability to bind DNA and to interact with RuvB and RecU. In spite of a high level of sequence conservation between RuvA(Mpn) and RuvA(Mge) (68.8% identity), substantial differences were found between these proteins in their activities. First, RuvA(Mge) was found to preferentially bind to HJs, whereas RuvA(Mpn) displayed similar affinities for both HJs and single-stranded DNA. Second, while RuvA(Mpn) is able to form two distinct complexes with HJs, RuvA(Mge) only produced a single HJ complex. Third, RuvA(Mge) stimulated the DNA helicase and ATPase activities of RuvB(Mge), whereas RuvA(Mpn) did not augment RuvB activity. Finally, while both RuvA(Mge) and RecU(Mge) efficiently bind to HJs, they did not compete with each other for HJ binding, but formed stable complexes with HJs over a wide protein concentration range. This interaction, however, resulted in inhibition of the HJ resolution activity of RecU(Mge).     

The N-Terminal Region of the RecU Holliday Junction Resolvase Is Essential for Homologous Recombination

The RecU Holliday junction (HJ)-resolving enzyme is highly conserved in the Firmicutes phylum of bacteria. In Bacillus subtilis, the recU gene has two putative initiation codons, at positions 1 and 33. In rec⁺ cells, only the full-length RecU polypeptide (206 residues, 23.9 kDa) was detected even after different stress treatments. To address the relevance of the flexible N-terminus, we constructed mutant variants. Experiments in vivo revealed that recUΔ1-32 (which initiates at Met33 and encodes RecUΔ1-32) and recU31 (the conserved Arg31 residue was substituted with alanine to give RecUR31A) are genuine RecU mutants, rendering cells impaired in DNA repair and chromosomal segregation. RecU has three activities: It (i) cleaves HJs, (ii) anneals complementary strands and (iii) modulates RecA activities. RecUR31A binds and cleaves HJ DNA in vitro as efficiently as wild-type RecU, but RuvB·ATPγS·Mg²⁺ fails to stimulate the RecUR31A cleavage reaction. In contrast, RecUΔ1-32 forms unstable complexes with DNA and fails to cleave HJs. RecU and its variants are capable of promoting DNA strand annealing and exert a negative effect on deoxy-ATP-dependent RecA-mediated DNA strand exchange. This study shows that the flexible N-terminus of RecU is essential for protein activity.     

Mutational analysis of endonuclease V from Thermotoga maritima

Endonuclease V nicks damaged DNA at the second phosphodiester bond 3' to inosine, uracil, mismatched bases, or abasic (AP) sites. Alanine scanning mutagenesis was performed in nine conserved positions of Thermotoga maritima endonuclease V to identify amino acid residues involved in recognition or endonucleolytic cleavage of these diverse substrates. Alanine substitution at D43, E89, and D110 either abolishes or substantially reduces inosine cleavage activity. These three mutants gain binding affinity for binding to double-stranded or single-stranded inosine substrates in the absence of a metal ion, suggesting that these residues may be involved in coordinating catalytic metal ion(s). Y80A, H116A, and, to a lesser extent, R88A demonstrate reduced affinities for double-stranded or single-stranded inosine substrates or nicked products. The lack of tight binding to a nicked inosine product accounts for the increased rate of turnover of inosine substrate since the product release is less rate-limiting. Y80A, R88A, and H116A fail to cleave AP site substrates. Their activities toward uracil substrates are in the following order: H116A > R88A > Y80A. These residues may play a role in substrate recognition. K139A maintains wild-type binding affinity for binding to double-stranded and single-stranded inosine substrate, but fails to cleave AP site and uracil substrate efficiently, suggesting that K139 may play a role in facilitating non-inosine substrate cleavage.     

The RecU Holliday junction resolvase acts at early stages of homologous recombination

Homologous recombination is essential for DNA repair and generation of genetic diversity in all organisms. It occurs through a series of presynaptic steps where the substrate is presented to the recombinase (RecA in bacteria). Then, the recombinase nucleoprotein filament mediates synapsis by first promoting the formation of a D-loop and later of a Holliday junction (HJ) that is subsequently cleaved by the HJ resolvase. The coordination of the synaptic step with the late resolution step is poorly understood. Bacillus subtilis RecU catalyzes resolution of HJs, and biochemical evidence suggests that it might modulate RecA. We report here the isolation and characterization of two mutants of RecU (recU56 and recU71), which promote resolution of HJs, but do not promote RecA modulation. In vitro, the RecU mutant proteins (RecUK56A or RecUR71A) bind and cleave HJs and interact with RuvB. RecU interacts with RecA and inhibits its single-stranded DNA-dependent dATP hydrolysis, but RecUK56A and RecUR71A do not exert a negative effect on the RecA dATPase and fail to interact with it. Both activities are important in vivo since RecU mutants impaired only in RecA interaction are as sensitive to DNA damaging agents as a deletion mutant.     

Truncation of the amino-terminus of the recombinant aggrecan rAgg1mut leads to reduced cleavage at the aggrecanase site. Efficient aggrecanase catabolism may depend on multiple substrate interactions.

Aggrecanase cleavage at the Glu(373)-Ala(374) site in the interglobular domain of the cartilage proteoglycan aggrecan is a key event in arthritic diseases. The observation that substrates representing only the aggrecanase cleavage site are not catabolized efficiently by aggrecanase prompted us to investigate the requirement of aggrecanase for additional structural elements of its substrate other than the actual cleavage site. Based on the recombinant substrate rAgg1mut we constructed deletion mutants with successively truncated N- or C-termini of the interglobular domain. Catabolism by aggrecanase activities induced in rat chondrosarcoma cells, porcine chondrocytes, and by human recombinant ADAMTS4 showed a gradually decreasing catabolism of progressively shortened, N-terminal deletion mutants of the substrate rAgg1mut. A reduction to 32 amino acids N-terminal to the aggrecanase site resulted in a decrease of at least 42% of aggrecanase cleavage products as compared with the wild-type substrate. When only 16 amino acids preceded the Glu(373)-Ala(374) site, aggrecanase cleavage was completely inhibited. In contrast, C-terminal deletions did not negatively affect aggrecanase cleavage up to the reduction to 13 amino acids C-terminal to the cleavage site. Unlike aggrecanase(s), membrane type 1-matrix metalloprotease (MT1-MMP), able to cleave rAgg1mut both at the aggrecanase and the MMP site, was insensitive to N-terminal deletions regarding aggrecanase cleavage, indicating that the importance of the N-terminus is characteristic for aggrecanase(s). Taken together, the results demonstrate that the amino-terminus of rAgg1mut, containing the MMP site, plays an important role for efficient cleavage by aggrecanase(s), possibly by serving as a further site of interaction between the enzyme and its substrate.     

Active site specificity of plasmepsin II.

Members of the aspartic proteinase family of enzymes have very similar three-dimensional structures and catalytic mechanisms. Each, however, has unique substrate specificity. These distinctions arise from variations in amino acid residues that line the active site subsites and interact with the side chains of the amino acids of the peptides that bind to the active site. To understand the unique binding preferences of plasmepsin II, an enzyme of the aspartic proteinase class from the malaria parasite, Plasmodium falciparum, chromogenic octapeptides having systematic substitutions at various positions in the sequence were analyzed. This enabled the design of new, improved substrates for this enzyme (Lys-Pro-Ile-Leu-Phe*Nph-Ala/Glu-Leu-Lys, where * indicates the cleavage point). Additionally, the crystal structure of plasmepsin II was analyzed to explain the binding characteristics. Specific amino acids (Met13, Ser77, and Ile287) that were suspected of contributing to active site binding and specificity were chosen for site-directed mutagenesis experiments. The Met13Glu and Ile287Glu single mutants and the Met13Glu/Ile287Glu double mutant gain the ability to cleave substrates containing Lys residues.     

Mutational and comparative analysis of streptolysin O, an oxygen-labile streptococcal hemolysin

The structural gene of streptolysin O was cloned from Streptococcus pyogenes strain Sa and S. equisimilis H46A, and the nucleotide sequences were compared with those of strain Richards. To obtain the minimal active fragment of the toxin and to elucidate structure-function relationships in hemolytic function, streptolysin O mutants deleted in N- and C-terminal regions were constructed. Internal amino acid residues were also replaced by introduction of point mutations. Analyses of these mutants showed that considerable activity was retained even after deletion of the N-terminal 107 residues, but genetic removal of the ultimate C-terminal residue resulted in a marked decrease in hemolytic function. By removal in succession, hemolytic activity declined exponentially, and only 0.002% of the activity remained after deletion of the C-terminal four residues. Nucleotide replacement experiments indicated pivotal roles of I202, V217, D324-L325, V339, and H469 residues in hemolysis.     

Identification of a C-terminal region that regulates mitogen-activated protein kinase kinase-1 cytoplasmic localization and ERK activation

The C-terminal region of mitogen-activated protein kinase kinase-1 and 2 (MKK1 and MKK2) may function in regulating interactions with upstream kinases or the magnitude and duration of ERK mitogen-activated protein kinase activity. The MKK C-terminal region contains a proline-rich region that reportedly functions in regulating interactions with the Raf-1 kinase and ERK activity. In addition, phosphorylation sites in the C terminus of MKK1 have been suggested to either sustain or attenuate MKK1 activity. To further understand how phosphorylation at the C terminus of MKK1 and protein interactions regulate MKK1 function, we have generated several MKK1 C-terminal deletion mutants and examined their function in regulating MKK1 localization, ERK protein activation, and cell growth. A deletion of C-terminal amino acids encompassing two putative alpha-helices between residues 330 and 379 caused a re-distribution of mutant MKK1 proteins to membrane compartments. Immunofluorescence analysis of MKK1 mutants revealed a loss of homogenous cytosolic distribution that is typically observed with MKK1 wild type, suggesting this region regulates MKK1 cellular localization. In contrast, MKK1 C-terminal deletion mutants localized to various sized punctate regions that overlapped with lysosome compartments. ERK activation in response to constitutively active Raf-1 or growth factor stimulus was attenuated in cells expressing MKK1 C-terminal deletion mutants. This could be partly explained by the inability of Raf-1 to phosphorylate MKK1 C-terminal deletion mutants even though the phosphorylation sites were intact in these mutants. Finally, we show that cells expressing MKK1 C-terminal deletion mutants displayed characteristic patterns of apoptotic cell death and reduced cell proliferation. These findings identify a novel C-terminal region between amino acid residues 330 and 379 on MKK1 that is necessary for regulating the cytoplasmic distribution and subsequent ERK protein activation necessary for cell survival and viability.     

Identification of Ligand Specificity Determinants in Lantibiotic Bovicin HJ50 and the Receptor BovK,a Multitransmembrane Histidine Kinase

Lantibiotic bovicin HJ50 is produced by Streptococcus bovis HJ50 and acts as the extracellular signal to autoregulate its own biosynthesis through BovK/R two-component system. Bovicin HJ50 shows a linear N-terminal and glubolar C-terminal structure, and the sensor histidine kinase BovK contains eight transmembrane segments lacking any extensive surface-exposed sensory domain. The signal recognition mechanism between bovicin HJ50 and BovK is still unknown. We performed saturated alanine scanning mutagenesis and other amino acid substitutions on bovicin HJ50 using a semi-in vitro biosynthesis. Results of the mutants inducing activities indicated that several charged and hydrophobic amino acids in ring B of bovicin HJ50, as well as two glycines were key residues to recognize BovK. Circular dichroism analyses indicated that both glycines contributed to bovicin HJ50 structural changes in the membrane. Biotin-labeled bovicin HJ50 could interact with the N-terminal sensor of BovK, and several charged residues and a conserved hydrophobic region in the N-terminal portion of BovK sensor domain were important for interacting with the signal bovicin HJ50. By combining the results, we suggested a mechanism of bovicin HJ50 recognizing and activating BovK mainly through electrostatic and hydrophobic interactions.     

Substrate recognition of VAMP-2 by botulinum neurotoxin B and tetanus neurotoxin

Botulinum neurotoxin (BoNT; serotypes A-G) and tetanus neurotoxin elicit flaccid and spastic paralysis, respectively. These neurotoxins are zinc proteases that cleave SNARE proteins to inhibit synaptic vesicle fusion to the plasma membrane. Although BoNT/B and tetanus neurotoxin (TeNT) cleave VAMP-2 at the same scissile bond, their mechanism(s) of VAMP-2 recognition is not clear. Mapping experiments showed that residues 60-87 of VAMP-2 were sufficient for efficient cleavage by BoNT/B and that residues 40-87 of VAMP-2 were sufficient for efficient TeNT cleavage. Alanine-scanning mutagenesis and kinetic analysis identified three regions within VAMP-2 that were recognized by BoNT/B and TeNT: residues adjacent to the site of scissile bond cleavage (cleavage region) and residues located within N-terminal and C-terminal regions relative to the cleavage region. Analysis of residues within the cleavage region showed that mutations at the P7, P4, P2, and P1' residues of VAMP-2 had the greatest inhibition of LC/B cleavage (> or =32-fold), whereas mutations at P7, P4, P1', and P2' residues of VAMP-2 had the greatest inhibition of LC/TeNT cleavage (> or =64-fold). Residues within the cleavage region influenced catalysis, whereas residues N-terminal and C-terminal to the cleavage region influenced binding affinity. Thus, BoNT/B and TeNT possess similar organization but have unique residues to recognize and cleave VAMP-2. These studies provide new insights into how the clostridial neurotoxins recognize their substrates.     

Shedding of collagen XVII/BP180: structural motifs influence cleavage from cell surface

Collagen XVII/BP180, an epithelial adhesion molecule, belongs to the group of collagenous transmembrane proteins, which are characterized by ectodomain shedding. We recently showed that ADAMs can cleave collagen XVII, but also that furin participates in this process (Franzke, C. W., Tasanen, K., Sch?cke, H., Zhou, Z., Tryggvason, K., Mauch, C., Zigrino, P., Sunnarborg, S., Lee, D. C., Fahrenholz, F., and Bruckner-Tuderman, L. (2002) EMBO J. 21, 5026-5035). To define the cleavage region in the juxtamembranous NC16A linker domain and assess its structure and requirements for shedding, we constructed deletion mutants of the NC16A domain, expressed them in COS-7 cells, and analyzed their structural integrity and shedding behavior. A mutant lacking the furin consensus sequence was shed in a normal manner, demonstrating that furin does not cleave collagen XVII but rather activates ADAMs (a disintegrin and metalloproteinase). Large deletions of the NC16A domain prevented shedding, and analysis of defined smaller deletions pointed to the stretch of amino acid residues 528-547 as important for sheddase recognition and cleavage. Secondary protein structure predictions showed that deletion of this stretch resulted in an NC16A domain with a positive net charge and an amphipathic alpha-helix, which can cause conformational changes in the collagen XVII homotrimer. Assessment of triple-helix folding of the mutants revealed a lower thermal stability of all non-shed variants than of wild-type collagen XVII or the shed mutants. In contrast, deletion of the putative nucleation site for triple-helix folding of collagenous transmembrane proteins did not affect folding of collagen XVII. The data indicate that the conformation of the NC16A domain and steric availability of the cleavage site influence shedding and is important for folding of collagen XVII.     

Inhibitory role of TACE/ADAM17 cytotail in protein ectodomain shedding

AIM:To determine if the cytotail of the principal sheddase tumor necrosis factor-α converting enzyme (TACE;ADAM17) controls protein ectodomain shedding.METHODS:Site-directed mutagenesis was performed to derive TACE variants. The resulting TACE expression plasmids with amino acid substitutions in the extracel-lular,cysteine-rich disintegrin domain (CRD) and/or deleted cytotail,along with an expression vector for the enhanced green fluorescence protein were transfected into shedding-defective M1 mutants stably expressing transmembrane L-selectin or transforming growth factor (TGF)-α. The expression levels of the TACE substrates at the cell surface were determined by flow cytometry. RESULTS:Consistent with published data,a single point mutation (C600Y) in the CRD led to shedding defi-ciency. However,removal of the cytotail from the C600Y TACE variant partially restored ectodomain cleavage of TGF-α and L-selectin. Cytotail-deleted mutants with any other substituting amino acid residues in place of Cys600 displayed similar function compared with tail-less C600Y TACE.CONCLUSION:The cytotail plays an inhibitory role,which becomes evident when it is removed from an enzyme with another mutation that affects the enzyme function.     

HA基因322位和329位氨基酸对H5N1亚型禽流感病毒毒力的影响

A/mallard/Huadong/S/2005(S,IVPI=2.65)和A/mallard/Huadong/Y/2003(Y,IVPI=O),是对麻鸭具有不同致病力的病毒.两病毒的HA裂解位点区有2个氨基酸差异,S病毒在HA裂解位点区322是Leu(L322),329位缺失(-329),而Y病毒322位是Gin(Q 322),329位是Lys(K329).根据这两个位点的差异,利用反向遗传系统,以S和Y病毒各自为骨架,拯救HA基因突变病毒,检测获救的突变病毒对麻鸭的毒力.可以得知,以S病毒为骨架,将S病毒HA基因322位Leu替换为Gln和(或)在329位添加Lys,以及用Y病毒的HA(Q322L,K329-)替换S病毒HA,获救的重组病毒对麻鸭亦完全无致病力;但以Y病毒为骨架,将Y病毒HA基因322位Gln替换为Leu和(或)在329位缺失Lys后,Y重组病毒对麻鸭的毒力上升.结果提示,S和Y病毒HA基因裂解位点区322和329氨基酸残基突变或缺失均影响病毒对麻鸭的致病力,且HA基因与其它基因的匹配性显著影响病毒对麻鸭的致病力.     

Identification and characterization of three members of the human metallocarboxypeptidase gene family

Amino acid homology searches of the human genome revealed three members of the metallocarboxypeptidase (metallo-CP) family that had not been described in the literature in addition to the 14 known genes. One of these three, named CPA5, is present in a gene cluster with CPA1, CPA2, and CPA4 on chromosome 7. The cDNA encoding a mouse homolog of human CPA5 was isolated from a testis library and sequenced. The deduced amino acid sequence of human CPA5 has highest amino acid sequence identity (60%) to CPA1. Modeling analysis shows the overall structure to be very similar to that of other members of the A/B subfamily of metallocarboxypeptidases. The active site of CPA5 is predicted to cleave substrates with C-terminal hydrophobic residues, as do CPA1, -2, and -3. Using Northern blot analysis, CPA5 mRNA is detected in testis but not in kidney, liver, brain, or lung. In situ hybridization analysis shows that CPA5 is localized to testis germ cells. Mouse pro-CPA5 protein expressed in Sf9 cells using the baculovirus system was retained in the particulate fraction of the cells and was not secreted into the media. Pro-CPA5 was not enzymatically active toward standard CPA substrates, but after incubation with prohormone convertase 4 the resulting protein was able to cleave furylacryloyl-Gly-Leu, with 3-4-fold greater activity at pH 7.4 than at 5.6. Two additional members of the human CP gene family were also studied. Modeling analysis indicates that both contain the necessary amino acids required for enzymatic activity. The CP on chromosome 8 is predicted to have a CPA-like specificity for C-terminal hydrophobic residues and was named CPA6. The CP on chromosome 2 is predicted to cleave substrates with C-terminal acidic residues and was named CPO.     

Identification of a discrete intermediate in the assembly/disassembly of physalis mottle tymovirus through mutational analysis.

Assembly intermediates of icosahedral viruses are usually transient and are difficult to identify. In the present investigation, site-specific and deletion mutants of the coat protein gene of physalis mottle tymovirus (PhMV) were used to delineate the role of specific amino acid residues in the assembly of the virus and to identify intermediates in this process. N-terminal 30, 34, 35 and 39 amino acid deletion and single C-terminal (N188) deletion mutant proteins of PhMV were expressed in Escherichia coli. Site-specific mutants H69A, C75A, W96A, D144N, D144N-T151A, K143E and N188A were also constructed and expressed. The mutant protein lacking 30 amino acid residues from the N terminus self-assembled to T=3 particles in vivo while deletions of 34, 35 and 39 amino acid residues resulted in the mutant proteins that were insoluble. Interestingly, the coat protein (pR PhCP) expressed using pRSET B vector with an additional 41 amino acid residues at the N terminus also assembled into T=3 particles that were more compact and had a smaller diameter. These results demonstrate that the amino-terminal segment is flexible and either the deletion or addition of amino acid residues at the N terminus does not affect T=3 capsid assembly. In contrast, the deletion of even a single residue from the C terminus (PhN188Delta1) resulted in capsids that were unstable. These capsids disassembled to a discrete intermediate with a sedimentation coefficent of 19.4 S. However, the replacement of C-terminal asparagine 188 by alanine led to the formation of stable capsids. The C75A and D144N mutant proteins also assembled into capsids that were as stable as the pR PhCP, suggesting that C75 and D144 are not crucial for the T=3 capsid assembly. pR PhW96A and pR PhD144N-T151A mutant proteins failed to form capsids and were present as heterogeneous aggregates. Interestingly, the pR PhK143E mutant protein behaved in a manner similar to the C-terminal deletion protein in forming unstable capsids. The intermediate with an s value of 19.4 S was the major assembly product of pR PhH69A mutant protein and could correspond to a 30mer. It is possible that the assembly or disassembly is arrested at a similar stage in pR PhN188Delta1, pR PhH69A and pR PhK143E mutant proteins.     

Mutational analysis of the channel and loop sequences of human ferritin H-chain.

Human ferritin H-chain mutants were obtained by engineering the recombinant protein expressed by Escherichia coli. The mutagenesis were directed to the C-terminal sequence forming the hydrophobic channel, to the hydrophilic channel and to the loop sequence. The mutants were analysed for extent of expression, for stability, for capacity to incorporate iron and for kinetics of iron uptake and iron oxidation. Of the 22 mutants analysed only two with deletions of single residues in the loop sequence and one with deletion of the last 28 amino acid residues did not assemble into ferritin-like proteins. The other mutants assembled correctly and showed similar chemical/physical properties to the wild-type; they included duplication of an 18-amino acid-residue stretch, deletion of the last 22 and the last seven residues and various mutations of single amino acid residues. Two mutants with extensive alteration in the C-terminal sequence had a diminished thermostability associated with incapability to incorporate iron though they still catalysed iron oxidation. The mutants with alterations of the sequence around the hydrophilic channel showed diminished iron uptake and oxidation kinetics, together with a slightly larger apparent molecular size. The results indicate (i) that two of the sequences are important for ferritin assembly/stability, (ii) that the presence of the hydrophobic channel is essential for formation of the iron core and (iii) that the sites of iron interaction and the path of iron penetration into ferritin remain unidentified.     

Functional analysis of crustacean Hyperglycemic Hormone by in vivo assay with wild-type and mutant recombinant proteins

The neuro-endocrine X-organ sinus-gland complex regulates important crustacean physiological processes, such as growth, reproduction and molting. Its major products are the neuropeptides of the cHH/MIH/GIH family. Until now the structure-function relationships of these neuropeptides were established by sequence comparison. To study the functional relevance of conserved amino acid residues or peptide motifs, we generated point and deletion mutants of the Norway lobster Nephrops norvegicus cHH. The wild type mature neuropeptide cHH and its mutant forms were expressed in bacteria as fusion proteins and assayed in vivo to assess their hyperglycemic activity. The wild type cHH had a hyperglycemic activity similar to that of cHH present in an eyestalk extract, and it was blocked by an anti-recombinant cHH antibody. Bioassays of cHHs, obtained by a progressive deletion of five highly conserved motifs, showed that the only deleted cHH, which conserves a hyperglycemic activity, is the one lacking the C-terminal motif, but still retaining all the motifs reported to be important for functional specificity and three-dimensional structure. All the cHH point mutants lacked a hyperglycemic activity. These results identify amino acid residues that are required for the hyperglycemic activity of cHH.     

Mutations in the interglobular domain of aggrecan alter matrix metalloproteinase and aggrecanase cleavage patterns. Evidence that matrix metalloproteinase cleavage interferes with aggrecanase activity

We have expressed G1-G2 mutants with amino acid changes at the DIPEN(341) downward arrow(342)FFGVG and ITEGE(373) downward arrow(374)ARGSV cleavage sites, in order to investigate the relationship between matrix metalloproteinase (MMP) and aggrecanase activities in the interglobular domain (IGD) of aggrecan. The mutation DIPEN(341) to DIGSA(341) partially blocked cleavage by MMP-13 and MMP-8 at the MMP site, while the mutation (342)FFGVG to (342)GTRVG completely blocked cleavage at this site by MMP-1, -2, -3, -7, -8, -9, -13, -14. Each of the MMP cleavage site mutants, including a four-amino acid deletion mutant lacking residues ENFF(343), were efficiently cleaved by aggrecanase, suggesting that the primary sequence at the MMP site had no effect on aggrecanase activity in the IGD. The mutation (374)ARGSV to (374)NVYSV completely blocked cleavage at the aggrecanase site by aggrecanase, MMP-8 and atrolysin C but had no effect on the ability of MMP-8 and MMP-13 to cleave at the Asn(341) downward arrowPhe bond. Susceptibility to atrolysin C cleavage at the MMP site was conferred in the DIGSA(341) mutant but absent in the wild-type, (342)GTRVG, (374)NVYSV, and deletion mutants. To further explore the relationship between MMP and aggrecanase activities, sequential digest experiments were done in which MMP degradation products were subsequently digested with aggrecanase and vice versa. Aggrecanase-derived G1 domains with ITEGE(373) C termini were viable substrates for MMPs; however, MMP-derived G2 fragments were resistant to cleavage by aggrecanase. A 10-mer peptide FVDIPENFFG, which is a substrate analogue for the MMP cleavage site, inhibited aggrecanase cleavage at the Glu(373) downward arrowAla bond. This study demonstrates that MMPs and aggrecanase have unique substrate recognition in the IGD of aggrecan and suggests that sequences at the C terminus of the DIPEN(341) G1 domain may be important for regulating aggrecanase cleavage.     

Engineering the active center of the 6-phospho-beta-galactosidase from Lactococcus lactis

Several amino acids in the active center of the 6-phospho-beta-galactosidase from Lactococcus lactis were replaced by the corresponding residues in homologous enzymes of glycosidase family 1 with different specificities. Three mutants, W429A, K435V/Y437F and S428D/ K435V/Y437F, were constructed. W429A was found to have an improved specificity for glucosides compared with the wild-type, consistent with the theory that the amino acid at this position is relevant for the distinction between galactosides and glucosides. The k(cat)/K(m) for o-nitrophenyl-beta-D-glucose-6-phosphate is 8-fold higher than for o-nitrophenyl-beta-D-galactose-6-phosphate which is the preferred substrate of the wild-type enzyme. This suggests that new hydrogen bonds are formed in the mutant between the active site residues, presumably Gln19 or Trp421 and the C-4 hydroxyl group. The two other mutants with the exchanges in the phosphate-binding loop were tested for their ability to bind phosphorylated substrates. The triple mutant is inactive. The double mutant has a dramatically decreased ability to bind o-nitrophenyl-beta-D-galactose-6-phosphate whereas the interaction with o-nitrophenyl-beta-D-galactose is barely altered. This result shows that the 6-phospho-beta-galactosidase and the related cyanogenic beta-glucosidase from Trifolium repens have different recognition mechanisms for substrates although the structures of the active sites are highly conserved.