N-linked keratan sulfate in the aggrecan interglobular domain potentiates aggrecanase activity

Keratan sulfate is thought to influence the cleavage of aggrecan by metalloenzymes. We have therefore produced a recombinant substrate, substituted with keratan sulfate, suitable for the study of aggrecanolysis in vitro. Recombinant human G1-G2 was produced in primary bovine keratocytes using a vaccinia virus expression system. Following purification and digestion with specific hydrolases, fluorophore-assisted carbohydrate electrophoresis was used to confirm the presence of the monosulfated Gal-GlcNAc6S and GlcNAc6s-Gal disaccharides and the disulfated Gal6S-GlcNAc6S disaccharides of keratan sulfate. Negligible amounts of fucose or sialic acid were detected, and the level of unsulfated disaccharides was minimal. Treatment with keratanases reduced the size of the recombinant G1-G2 by approximately 5 kDa on SDS-PAGE. Treatment with N-glycosidase F also reduced the size of G1-G2 by approximately 5 kDa and substantially reduced G1-G2 immunoreactivity with monoclonal antibody 5-D-4, indicating that keratan sulfate on the recombinant protein is N-linked. Cleavage of G1-G2 by aggrecanase was markedly reduced when keratan sulfate chains were removed by treatment with keratanase, keratanase II, endo-beta-galactosidase, or N-glycosidase F. These results indicate that modification of oligosaccharides in the aggrecan interglobular domain with keratan sulfate, most likely at asparagine residue 368, potentiates aggrecanase activity in this part of the core protein.     

Susceptibility to ozone-induced acute lung injury in iNOS-deficient mice

Mice deficient in inducible nitric oxide synthase (iNOS; C57Bl/6Ai-[KO]NOS2 N5) or wild-type C57Bl/6 mice were exposed to 1 part/million of ozone 8 h/night or to filtered air for three consecutive nights. Endpoints measured included lavagable total protein, macrophage inflammatory protein (MIP)-2, matrix metalloproteinase (MMP)-9, cell content, and tyrosine nitration of whole lung proteins. Ozone exposure caused acute edema and an inflammatory response in the lungs of wild-type mice, as indicated by significant increases in lavage protein content, MIP-2 and MMP-9 content, and polymorphonuclear leukocytes. The iNOS knockout mice showed significantly greater levels of lung injury by all of these criteria than did the wild-type mice. We conclude that iNOS knockout mice are more susceptible to acute lung damage induced by exposure to ozone than are wild-type C57Bl/6 mice and that protein nitration is associated with the degree of inflammation and not dependent on iNOS-derived nitric oxide.     

Arabidopsis UVR8 regulates ultraviolet-B signal transduction and tolerance and contains sequence similarity to human regulator of chromatin condensation 1

To further our understanding of how plants defend against the harmful effects of ultraviolet (UV) light, we characterized an Arabidopsis mutant hypersensitive to UV-B. This mutant, UV resistance locus 8-1 (uvr8-1), contains a single recessive mutation at the bottom of chromosome 5. Fine-scale mapping localized uvr8-1 to a 21-kb locus containing five predicted open reading frames. Sequencing of this entire region revealed that the uvr8-1 allele contains a 15-nucleotide deletion in a gene similar to the human guanine nucleotide exchange factor regulator of chromatin condensation 1. This mutation reduces the UV-B-mediated induction of flavonoids and blocks chalcone synthase mRNA and protein induction. In contrast, uvr8-1 has enhanced induction of PR1 and PR5 proteins in response to UV-B, an indication of increased UV-B injury. These results suggest that UVR8 acts in a UV-B signal transduction pathway leading to induction of flavonoid biosynthesis.     

Ethylmethanesulfonate saturation mutagenesis in Arabidopsis to determine frequency of herbicide resistance

Plant resistance to glyphosate has been reported far less frequently than resistance to sulfonylurea and imidazolinone herbicides. However, these studies tend to be anecdotal, without side by side comparisons for a single species or natural isolate. In this study, we tested the frequencies of resistance of three herbicides in a controlled ethylmethanesulfonate (EMS) saturation mutagenesis experiment, allowing a direct comparison of the frequencies at which resistant mutant plants arise. The 100% growth inhibition dose rates of glyphosate, chlorsulfuron (a sulfonylurea herbicide), and imazethapyr (an imidazolinone herbicide) were determined for Arabidopsis. Populations of EMS-mutagenized M(2) seedlings were sprayed with twice the 100% growth inhibition dose of glyphosate, chlorsulfuron, or imazethapyr, and herbicide-resistant mutants were identified. Although there were no glyphosate-resistant mutants among M(2) progeny of 125,000 Columbia and 125,000 Landsberg erecta M(1) lines, chlorsulfuron resistance and imazethapyr resistance each appeared at frequencies of 3.2 x 10(-5). Given the observed frequency of herbicide resistance mutations, we calculate that there are at least 700 mutations in each EMS-mutagenized Arabidopsis line and that fewer than 50,000 M(1) lines are needed to have a 95% chance of finding a mutation in any given G:C base pair in the genome. As part of this study, two previously unreported Arabidopsis mutations conferring resistance to imidazolinone herbicides, csr1-5 (Ala-122-Thr) and csr1-6 (Ala-205-Val), were discovered. Neither of these mutations caused enhanced resistance to chlorsulfuron in Arabidopsis.     

Engineering vitamin E content: from Arabidopsis mutant to soy oil

We report the identification and biotechnological utility of a plant gene encoding the tocopherol (vitamin E) biosynthetic enzyme 2-methyl-6-phytylbenzoquinol methyltransferase. This gene was identified by map-based cloning of the Arabidopsis mutation vitamin E pathway gene3-1 (vte3-1), which causes increased accumulation of delta-tocopherol and decreased gamma-tocopherol in the seed. Enzyme assays of recombinant protein supported the hypothesis that At-VTE3 encodes a 2-methyl-6-phytylbenzoquinol methyltransferase. Seed-specific expression of At-VTE3 in transgenic soybean reduced seed delta-tocopherol from 20 to 2%. These results confirm that At-VTE3 protein catalyzes the methylation of 2-methyl-6-phytylbenzoquinol in planta and show the utility of this gene in altering soybean tocopherol composition. When At-VTE3 was coexpressed with At-VTE4 (gamma-tocopherol methyltransferase) in soybean, the seed accumulated to >95% alpha-tocopherol, a dramatic change from the normal 10%, resulting in a greater than eightfold increase of alpha-tocopherol and an up to fivefold increase in seed vitamin E activity. These findings demonstrate the utility of a gene identified in Arabidopsis to alter the tocopherol composition of commercial seed oils, a result with both nutritional and food quality implications.     

ADAMTS4 cleaves at the aggrecanase site (Glu373-Ala374) and secondarily at the matrix metalloproteinase site (Asn341-Phe342) in the aggrecan interglobular domain

Two major proteolytic cleavages, one at NITEGE(373)/A(374)RGSVI and the other at VDIPEN(341)/F(342)FGVGG, have been shown to occur in vivo within the interglobular domain of aggrecan. The Glu(373)-Ala(374) site is cleaved in vitro by aggrecanase-1 (ADAMTS4) and aggrecanase-2 (ADAMTS5), whereas the other site, at Asn(341)-Phe(342), is efficiently cleaved by matrix metalloproteinases (MMPs) and by cathepsin B at low pH. Accordingly, the presence of the cleavage products globular domain 1 (G1)-NITEGE(373) and G1-VDIPEN(341) in vivo has been widely interpreted as evidence for the specific involvement of ADAMTS enzymes and MMPs/cathepsin B, respectively, in aggrecan proteolysis in situ. We show here, in digests with native human aggrecan, that purified ADAMTS4 cleaves primarily at the Glu(373)-Ala(374) site, but also, albeit slowly and secondarily, at the Asn(341)-Phe(342) site. Cleavage at the Asn(341)-Phe(342) site in these incubations was due to bona fide ADAMTS4 activity (and not a contaminating MMP) because the cleavage was inhibited by TIMP-3 (a potent inhibitor of ADAMTS4), but not by TIMP-1 and TIMP-2, at concentrations that totally blocked MMP-3-mediated cleavage at this site. Digestion of recombinant human G1-G2 (wild-type and cleavage site mutants) confirmed the dual activity of ADAMTS4 and supported the idea that the enzyme cleaves primarily at the Glu(373)-Ala(374) site and secondarily generates G1-VDIPEN(341) by removal of the Phe(342)-Glu(373) peptide from G1-NITEGE(373). These results show that G1-VDIPEN(341) is a product of both MMP and ADAMTS4 activities and challenge the widely held assumption that this product represents a specific indicator of MMP- or cathepsin B-mediated aggrecan degradation.     

Paired sequence difference in ribosomal RNAs: evolutionary and phylogenetic implications

Ribosomal RNAs have secondary structures that are maintained by internal Watson-Crick pairing. Through analysis of chordate, arthropod, and plant 5S ribosomal RNA sequences, we show that Darwinian selection operates on these nucleotide sequences to maintain functionally important secondary structure. Insect phylogenies based on nucleotide positions involved in pairing and the production of secondary structure are incongruent with those constructed on the basis of positions that are not. Furthermore, phylogeny reconstruction using these nonpairing bases is concordant with other, morphological data.      

Suppressors of trp1 fluorescence identify a new arabidopsis gene, TRP4, encoding the anthranilate synthase beta subunit.

Suppressors of the blue fluorescence phenotype of the Arabidopsis trp1-100 mutant can be used to identify mutations in genes involved in plant tryptophan biosynthesis. Two recessive suppressor mutations define a new gene, TRP4. The trp4 mutant and the trp1-100 mutant are morphologically normal and grow without tryptophan, whereas the trp4; trp1-100 double mutant requires tryptophan for growth. The trp4; trp1-100 double mutant does not segregate at expected frequencies in genetic crosses because of a female-specific defect in transmission of the double mutant genotype, suggesting a role for the tryptophan pathway in female gametophyte development. Genetic and biochemical evidence shows that trp4 mutants are defective in a gene encoding the beta subunit of anthranilate synthase (AS). Arabidopsis AS beta subunit genes were isolated by complementation of an Escherichia coli anthranilate synthase mutation. The trp4 mutation cosegregates with one of the genes, ASB1, located on chromosome 1. Sequence analysis of the ASB1 gene from trp4-1 and trp4-2 plants revealed different single base pair substitutions relative to the wild type. Anthranilate synthase alpha and beta subunit genes are regulated coordinately in response to bacterial pathogen infiltration.