The thrombospondin motif of aggrecanase-1 (ADAMTS-4) is critical for aggrecan substrate recognition and cleavage

Aggrecanase-1 (ADAMTS-4) is a member of the a disintegrin and metalloprotease with thrombospondin motifs (ADAMTS) protein family that was recently identified. Aggrecanase-1 is one of two ADAMTS cartilage-degrading enzymes purified from interleukin-1-stimulated bovine nasal cartilage (Tortorella, M. D., Burn, T. C., Pratta, M. A. , Abbaszade, I., Hollis, J. M., Liu, R., Rosenfeld, S. A., Copeland, R. A., Decicco, C. P., Wynn, R., Rockwell, A., Yang, F., Duke, J. L., Solomon, K., George, H., Bruckner, R., Nagase, H., Itoh, Y., Ellis, D. M., Ross, H., Wiswall, B. H., Murphy, K., Hillman, M. C., Jr., Hollis, G. F., and Arner, E.C. (1999) Science 284, 1664-1666; 2 Abbaszade, I., Liu, R. Q., Yang, F., Rosenfeld, S. A., Ross, O. H., Link, J. R., Ellis, D. M., Tortorella, M. D., Pratta, M. A., Hollis, J. M., Wynn, R., Duke, J. L., George, H. J., Hillman, M. C., Jr., Murphy, K., Wiswall, B. H., Copeland, R. A., Decicco, C. P., Bruckner, R., Nagase, H., Itoh, Y., Newton, R. C., Magolda, R. L., Trzaskos, J. M., and Burn, T. C. (1999) J. Biol. Chem. 274, 23443-23450). The aggrecan products generated by this enzyme are found in cartilage cultures stimulated with cytokines and in synovial fluid from patients with arthritis, suggesting that aggrecanase-1 may be important in diseases involving cartilage destruction. Here we demonstrate that the thrombospondin type-1 (TSP-1) motif located within the C terminus of aggrecanase-1 binds to the glycosaminoglycans of aggrecan. Data from several studies indicate that this binding of aggrecanase-1 to aggrecan through the TSP-1 motif is necessary for enzymatic cleavage of aggrecan. 1) A truncated form of aggrecanase-1 lacking the TSP-1 motif was not effective in cleaving aggrecan. 2) Several peptides representing different regions of the TSP-1 motif effectively blocked aggrecanase-1 cleavage of aggrecan by preventing the enzyme from binding to the substrate. 3) Aggrecanase-1 was not effective in cleaving glycosaminoglycan-free aggrecan. Taken together, these data suggest that the TSP-1 motif of aggrecanase-1 is critical for substrate recognition and cleavage.     

Assignment of the aliphatic 1H and 13C resonances of the Bacillus subtilis glucose permease IIA domain using double- and triple-resonance heteronuclear three-dimensional NMR spectroscopy.

Nearly complete assignment of the aliphatic 1H and 13C resonances of the IIAglc domain of Bacillus subtilis has been achieved using a combination of double- and triple-resonance three-dimensional (3D) NMR experiments. A constant-time 3D triple-resonance HCA(CO)N experiment, which correlates the 1H alpha and 13C alpha chemical shifts of one residue with the amide 15N chemical shift of the following residue, was used to obtain sequence-specific assignments of the 13C alpha resonances. The 1H alpha and amide 15N chemical shifts had been sequentially assigned previously using principally 3D 1H-15N NOESY-HMQC and TOCSY-HMQC experiments [Fairbrother, W. J., Cavanagh, J., Dyson, H. J., Palmer, A. G., III, Sutrina, S. L., Reizer, J., Saier, M. H., Jr., & Wright, P. E. (1991) Biochemistry 30, 6896-6907]. The side-chain spin systems were identified using 3D HCCH-COSY and HCCH-TOCSY spectra and were assigned sequentially on the basis of their 1H alpha and 13C alpha chemical shifts. The 3D HCCH and HCA(CO)N experiments rely on large heteronuclear one-bond J couplings for coherence transfers and therefore offer a considerable advantage over conventional 1H-1H correlation experiments that rely on 1H-1H 3J couplings, which, for proteins the size of IIAglc (17.4 kDa), may be significantly smaller than the 1H line widths. The assignments reported herein are essential for the determination of the high-resolution solution structure of the IIAglc domain of B. subtilis using 3D and 4D heteronuclear edited NOESY experiments; these assignments have been used to analyze 3D 1H-15N NOESY-HMQC and 1H-13C NOESY-HSQC spectra and calculate a low-resolution structure [Fairbrother, W. J., Gippert, G. P., Reizer, J., Saier, M. H., Jr., & Wright, P. E. (1992) FEBS Lett. 296, 148-152].     

Construction of yeast strains containing genetically marked transposons

Haploid yeast cells contain approximately 35 Ty (transposon yeast) elements. To facilitate the study of these elements, we have constructed yeast strains in which one of these transposons carries a genetic marker. The elements that we have modified are Ty912 and Ty917, elements originally detected at the HIS4 locus in spontaneously occurring his4- mutants. The strain constructions took place in three stages: 1) cloning of the mutant HIS4 genes containing the Ty elements; 2) introduction of a HindIII restriction fragment containing the yeast URA3 gene into the cloned transposons; and 3) replacement of the chromosomal HIS4 gene with the modified genes constructed in vitro. These strains will be extremely useful in the study of Ty transposition and other Ty-promoted DNA rearrangements.     

Recent advances in the molecular analysis of inherited disease

Many important human genes have been cloned during the last ten years. In some cases, using reverse genetic techniques [Orkin, S. H. (1986) Cell 47, 845-850], disease-causing genes have been isolated whose product was previously unknown. Important examples include the dystrophin protein which, when mutated, gives rise to either Duchenne or Becker muscular dystrophy [Koenig, M., Hoffman, E. P., Bertelson, C. J., Monaco, A. P., Feener, C. and Kunkel, L. M. (1987) Cell 50, 509-517; Monaco, A. P., Bertelson, C. J., Liechti-Gallati, S. & Kunkel, L. M. (1988) Genomics 2, 90-95; Koenig, M., Monaco, A. P. & Kunkel, L. M. (1988) Cell 53, 219-228] and the cystic fibrosis transmembrane conductance regulator (CFTR) [Riordan, J. R., Rommens, J. M., Kerem, B.-S., Alon, N., Rozmahel, R., Grzelczak, Z., Zielenski, J., Lok, S., Plavsic, N., Chou, J.-L., Drumm, M. L., Ianuzzi, M. C., Collins, F. S. & Tsui, L.-C. (1989) Science 245, 1066-1073]. Recently the technology for systematically detecting single base-pair changes by chemical methods, enzymatic methods or direct DNA sequencing has greatly expanded and simplified. In addition to providing structural information about these clinically important genes and information on disease-causing mutations, these studies have led to an increased understanding of mechanisms of mutation, to the discovery of novel genetic mechanisms and to important clinical applications of carrier detection and pre-natal diagnosis. The recent rapid progress has been made possible by the development of DNA amplification using the polymerase chain reaction (pcr) invented by Saiki and colleagues [Saiki, R. K., Chang, C-A., Levenson, C. H., Warren, T. C., Boehm, C. D., Kazazian, H. H. & Ehrlich, H. A. (1988) N. Engl. J. Med. 319, 537-541].     

Buchbesprechungen / Book Reviews

Book reviewed in this articles:
Sinclair, J. B. (Herausgeber) , Compendium of Soybean Diseases, 2nd edition.
Bach, W., J. Pankrath, S. H. Schneider (Herausgeber), Food-Climate Inter-actions.
Palti, J. , Cultural Practices and Infectious Crop Diseases.
Finck, A. , Fertilizers and Fertilization.
Turner, P. D. , Oil palm diseases and disorders.
Watkins, G. M. (ed.) , Compendium of cotton diseases.
Webster, H. S., Jr. , Plant Protection.
Robinson, D. G., and H. Quader (Ed.) , Cell Walls '81.
Progress in Botany (Fortschritte der Botanik) . Morphology — Physiology — Genetics — Taxonomy — Geobotany.
Maggenti, A. , General Nematology.
Chapeville, F., and A. L. Haenni (Ed.) , Chemical Recognition in Biology.
Rhodes-Roberts, M., und F. A. Skinner , Bacteria and Plants.
Michel, H,-G., und H. Umgelter , Pflanzenschutz im Garten.     

Mode of action and substrate specificities of cellulases from cloned bacterial genes

Three recombinant plasmids, pEC1, pEC2, and pEC3, each containing a unique Cellulomonas fimi chromosomal DNA insert, expressed Cm-cellulase activities in Escherichia coli C600 (Whittle, D. J., Kilburn, D. H., Warren, R. A. J., and Miller, R. C., Jr. (1982) Gene (Amst.) 17, 139-145; Gilkes, N. R., Kilburn, D. G., Langsford, M. L., Miller, R. C., Jr., Wakarchuk, W. W., Warren, R. A. J., Whittle, D. J., and Wong, W. K. R. (1984) J. Gen. Microbiol. 130, 1377-1384). Viscometric and chemical analyses showed that the enzymes encoded by pEC2 and pEC3 behaved as endoglucanases, whereas that encoded by pEC1 behaved as an exoglucanase. The activities of the exoglucanase and the pEC2-encoded endodglucanase were additive on Cm-cellulose as substrate. The pEC1-encoded enzyme also hydrolyzed xylan and p-nitrophenyl cellobioside. Two substrate-bound Cm-cellulases were isolated from the residual cellulose in a C. fimi culture by guanidine hydrochloride elution, affinity chromatography, and polyacrylamide gel electrophoresis. Both were glycoproteins of apparent Mr = 58,000 and 56,000, respectively. The 56-kDa enzyme appeared to be identical with the pEC1-encoded product, suggesting that they arise from the same gene.     

Comparison of the data, analysis, and results of X-ray absorption studies of cytochrome c oxidase

Differences in the methods of analysis of X-ray absorption data used by Powers et al. [Powers, L., Blumberg, W. E., Chance, B., Barlow, C., Leigh, J., Jr., Smith, J., Yonetani, T., Vik, S., & Peisach, J. (1979) Biochim. Biophys. Acta 547, 520-538; Powers, L., Chance, B., Ching, Y., & Angiolillo, P. (1981) Biophys. J. 34, 465-498] and Scott et al. [Scott, R., Schwartz, J., & Cramer S. (1986) Biochemistry 25, 5546-5555] are clarified. In addition, we compare the X-ray absorption data and results for resting cytochrome c oxidase reported by both groups using the same analysis method and conclude apart from any assumptions that the data are not identical.     

Recombination between retrotransposons as a source of chromosome rearrangements in the yeast Saccharomyces cerevisiae

Homologous recombination between dispersed repeated genetic elements is an important source of genetic variation. In this review, we discuss chromosome rearrangements that are a consequence of homologous recombination between transposable elements in the yeast Saccharomyces cerevisiae. The review will be divided into five sections: (1) Introduction (mechanisms of homologous recombination involving ectopic repeats), (2) Spontaneous chromosome rearrangements in wild-type yeast cells, (3) Chromosome rearrangements induced by low DNA polymerase, mutagenic agents or mutations in genes affecting genome stability, (4) Recombination between retrotransposons as a mechanism of genome evolution, and (5) Important unanswered questions about homologous recombination between retrotransposons. This review complements several others [S. Liebman, S. Picologlou, Recombination associated with yeast retrotransposons, in: Y. Koltin, M.J. Leibowitz (Eds.), Viruses of Fungi and Simple Eukaryotes, Marcel Dekker Inc., New York, 1988, pp. 63-89; P. Lesage, A.L. Todeschini, Happy together: the life and times of Ty retrotransposons and their hosts, Cytogenet. Genome Res. 110 (2005) 70-90; D.J. Garfinkel, Genome evolution mediated by Ty elements in Saccharomyces, Cytogenet. Genome Res. 110 (2005) 63-69] that discuss genomic rearrangements involving Ty elements.     

Heterogeneous Functional Ty1 Elements Are Abundant in the Saccharomyces Cerevisiae Genome

Despite the abundance of Ty1 RNA in Saccharomyces cerevisiae, Ty1 retrotransposition is a rare event. To determine whether transpositional dormancy is the result of defective Ty1 elements, functional and defective alleles of the retrotransposon in the yeast genome were quantitated. Genomic Ty1 elements were isolated by gap repair-mediated recombination of pGTy1-H3(Δ475-3944)HIS3, a multicopy plasmid containing a GAL1/Ty1-H3 fusion element lacking most of the gag domain (TYA) and the protease (PR) and integrase (IN) domains. Of 39 independent gap repaired pGTyHIS3 elements isolated, 29 (74%) transposed at high levels following galactose induction. The presence of restriction site polymorphisms within the gap repaired region of the 29 functional pGTyHIS3 elements indicated that they were derived from at least eight different genomic Ty1 elements and one Ty2 element. Of the 10 defective pGTyHIS3 elements, one was a partial gap repair event while the other nine were derived from at least six different genomic Ty1 elements. These results suggest that most genomic Ty1 elements encode functional TYA, PR and IN proteins. To understand how functional Ty1 elements are regulated, we tested the hypothesis that a TYB protein associates preferentially in cis with the RNA template that encodes it, thereby promoting transposition of its own element. A genomic Ty1 mhis3AI element containing either an in-frame insertion in PR or a deletion in TYB transposed at the same rate as a wild-type Ty1mhis3AI allele, indicating that TYB proteins act efficiently in trans. This result suggests in principle that defective genomic Ty1 elements could encode trans-acting repressors of transposition; however, expression of only one of the nine defective pGTy1 isolates had a negative effect on genomic Ty1 mhis3AI element transposition in trans, and this effect was modest. Therefore, the few defective Ty1 elements in the genome are not responsible for transpositional dormancy.     

Monte Carlo studies of phospholipid lamellae. Effects of proteins, cholesterol, bilayer curvature, and lateral mobility on order parameters

In this paper we present the results of a Monte Carlo study of the effects of protein, cholesterol, bilayer curvature, and mobility on the chain order parameters of a lipid layer. The Monte Carlo method used is identical to the version developed earlier (Scott, Jr., H.L. (1977) Biochim. Biophys. Acta 469, 264–271). Simulations of protein and cholesterol effects are accomplished by insertion of a rigid stationary cylinder into the lipid matrix. The protein studies show the presence of boundary lipid (Jost, P., Griffith, O.H., Capaldi, R.H. and Vanderkooi, G. (1973) Biochim. Biophys. Acta 311, 141–152). The effect of cholesterol is dependent upon the length of the lipid hydrocarbon chains relative to the cholesterol depth of penetration. Our computer studies of bilayer curvature show the manner in which this curvature disrupts chain packing and are consistent with experimental results (Chrzeszczyk, A., Wishnia, A. and Springer, C.S. (1977) Biochim. Biophys. Acta, 470, 161–171). We also find that restricting lateral motion in chains, the simplest manner in which head group interactions can affect hydrocarbon chain order, does not measurably alter the order parameters. We argue that this provides some support for an earlier hypothesis by Scott (Scott, Jr., H.L. (1975) Biochim. Biophys. Acta 406, 329–346) regarding head group-chain interaction in monolayer experiments.     

Binding of D-phenylalanine and D-tyrosine to carboxypeptidase A

The structures of the complexes of carboxypeptidase A with the amino acids D-phenylalanine and D-tyrosine are reported as determined by x-ray crystallographic methods to a resolution of 2.0 A. In each individual study one molecule of amino acids binds to the enzyme in the COOH-terminal hydrophobic pocket: the carboxylate of the bound ligand salt links with Arg-145, and the alpha-amino group salt links with Glu-270. The carboxylate of Glu-270 must break its hydrogen bond with the native zinc-bound water molecule in order to exploit the latter interaction. This result is in accord with spectroscopic studies which indicate that the binding of D or L amino acids (or analogues thereof) allows for more facile displacement of the metal-bound water by anions (Bicknell, R., Schaffer, A., Bertini, I., Luchinat, C., Vallee, B. L., and Auld, D. S. (1988) Biochemistry 27, 1050-1057). Additionally, we observe a significant movement of the zinc-bound water molecule (approximately 1 A) upon the binding of D-ligands. We propose that this unanticipated movement also contributes to anion sensitivity. The structural results of the current x-ray study correct predictions made in an early model building study regarding the binding of D-phenylalanine (Lipscomb, W. N., Hartsuck, J. A., Reeke, G. N., Jr., Quiocho, F. A., Bethge, P. H., Ludwig, M. L., Steitz, T. A., Muirhead, H., and Coppola, J. C. (1968) Brookhaven Symp. Biol. 21, 24-90).     

Intrachromosomal movement of genetically marked Saccharomyces cerevisiae transposons by gene conversion.

In this paper, we describe the movement of a genetically marked Saccharomyces cerevisiae transposon. Ty912(URA3), to new sites in the S. cerevisiae genome. Ty912 is an element present at the HIS4 locus in the his4-912 mutant. To detect movement of Ty912, this element has been genetically marked with the S. cerevisiae URA3 gene. Movement of Ty912(URA3) occurs by recombination between the marked element and homologous Ty elements elsewhere in the S. cerevisiae genome. Ty912(URA3) recombines most often with elements near the HIS4 locus on chromosome III, less often with Ty elements elsewhere on chromosome III, and least often with Ty elements on other chromosomes. These recombination events result in changes in the number of Ty elements present in the cell and in duplications and deletions of unique sequence DNA.     

Two immunologically and developmentally distinct chondroitin sulfate proteolglycans in embryonic chick brain.

Two different chondroitin sulfate proteoglycans (CSPG) in embryonic chick brain were distinguished by immunoreactivity either with S103L, a rat monoclonal antibody which reacts specifically with an 11-amino-acid region in the chondroitin sulfate domain of the core protein of chick cartilage CSPG (Krueger, R. C., Jr., Fields, T. A., Mensch, J. R., and Schwartz, N. B. (1990) J. Biol. Chem. 265, 12088-12097), or with HNK-1, a mouse monoclonal antibody which reacts with a 3-sulfoglucuronic acid residue on neural glycolipids and glycoproteins (Chou, D. K. H., Ilyas, A., Evans, J. E. Costello, C., Quarles, R. H., and Jungawala, F. B. (1986) J. Biol. Chem. 261, 11717-11725) but not with both antibodies. This specific immunoreactivity was used to separate the two CSPGs for further characterization. The S103L reactive brain proteoglycan had a core protein of similar size to cartilage CSPG (370 kDa) but exhibited a smaller hydrodynamic size (K(av) of 0.308). It was substituted predominantly with chondroitin sulfate chains and virtually no keratan sulfate chains. The HNK-1 reactive CSPG had a smaller core protein (340 kDa), an even smaller hydrodynamic size (K(av) of 0.564), and was substituted with both chondroitin sulfate and keratan sulfate chains. Glycosidase digestion patterns with endo-beta-galactosidase, N-glycosidase F, neuraminidase, and O-glycosidase, and reactivity with an antibody to the hyaluronate binding region also showed significant differences between the two brain CSPGs. Expression of the S103L reactive brain CSPG was developmentally regulated from embryonic day 7 through 19 with a peak in core protein on day 13, and in mRNA expression at day 10. In contrast the HNK-1 reactive brain CSPG was constitutively present from day 7 through hatching. These data suggest that these two distinct core proteins are immunologically and biochemically unique translation products of two different CSPG genes.