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1. Risk of Bowel Obstruction in Patients Undergoing Neoadjuvant Chemotherapy for High-risk Colon Cancer: A Nested Case-control Matched Analysis of an International, Multi-centre, Randomised Controlled Trial (FOxTROT)

Author

Glasbey, James, Glasbey, James, Beggs, Andrew, Glimelius, Bengt, Gray, Richard, Handley, Kelly, Laurberg, Søren, Magill, Laura, Murakami, Keigo, Palmer, Andy, Quirke, Philip, Seligman, Jenny, Seymour, Matt, Sinha, Yash, West, Nick, Morton, Dion, Glasbey, James, Handley, Kelly, Palmer, Andy, Morton, Dion, Crosby, T., Olliff, J., Peto (Chair), R., Brown, Gina, Ferry, David, Glimelius, Bengt, Gray, Richard, Handley, Kelly, Ismail, Tariq, Laurberg, Søren, Magill, Laura, Morton, Dion, Oliver, Alf, Quirke, Phil, Seymour, Matt, Scott, Nigel, Seligman, Jenny, Swift, Ian, Warren, Bryan, West, Nick, Northover, J., Parmar (Chair), M., Slevin, M., Magill, Laura, Gray, Richard, Handley, Kelly, Wilcockson, Adrian, Gray, Zoe, Lancaster, Dominic, Brown, James, Palmer, Andrew, Adie, Ladan, Kennedy, Georgia, Eld, M., Holt, G., Yilmaz, M., Spendler, K. Garm, Hansen, F., Laurberg, S., Rosenkilde, M., Ahlstrom, H., Glimelius, B., Abgamu, D., Day, N., Walsh, C., Bannister, J., Furniss, D., Morgan, S., Walkington, L., Yates, S., Branagan, G., Mustajab, A., O’Neil, H., Rees, C., Geh, I., Hendrickse, C., Langman, G., Pallan, A., Conn, A., Lowe, A., Ostrowski, J., Steward, M., Callaway, M., Falk, S., Thomas, M., Wong, N., Cast, J., Hartley, J., Roy, R., Tiam, R., Blunt, D., Cleator, S., Dawson, P., Goldin, R., Gujral, D., Lowdell, C., Ziprin, P., Clenton, S., Dewdney, A., Euinton, H., Furniss, D., Gupta, R., Tarapowewalla, D., Wilshaw, V., Braun, M., Chakrabarty, B., Hill, J., Laasch, H., Saunders, M., Cruickshank, N., Davies, M., Muzaffar, S., Orme, A., Punia, P., Rea, D., Campbell, F., Hughes, M., Palmer, D., Rooney, P., Abbott, G., Hamid, B., Vimalachandran, D., Berry, J., Hinson, F., Maarouf, Z., Nicoll, J., Adams, C., Denson, J., Jackson, S., Sherriff, D., Kweka, E., McAdam, G., Peters, M., Roy, R., Khaira, M., Kurien, G., Robinson, J., Wadsley, J., White, D., Young, R., Dega, R., Lamparelli, M., Orbell, J., Osborne, R., Taylor, P., Thomas, T., Gopalakrishnan, K., Jadhav, V., Scott-Brown, M., Baijal, S., Chapman, M., Glaholm, J., Nelson, C., Singh, R., Harrison, J., Last, K., Scott, D., Scullion, D., Lind, P., Milosavljevic, Z., Dent, J., Ilsley, D., Littleford, S., Roberts, C., Crabtree, M., Orrell, J., Sherwin, E., Smith, S., Soomal, R., Braun, M., De, A., Khan, A., Khan, U., Lavin, V., McBain, C., Radharkrishna, G., Sil, R., Weerasinghe, S., Hill, J., Lee, S., Wright, P., Church, R., Holland, C., Kunene, V., Thompson, A., Glynne-Jones, R., Goh, V., Livingstone, J., Richman, P., Barlow, C., Burn, P., Geraghty, J., Walther, J., Grumett, S., Mangalika, S., Qaiyum, M., Williams, G., Borgstein, R., Bridgewater, J., Melville, D., Rees, J., Coxon, F., Hainsworth, P., Needham, S., Scott, J., Asmussen, J., Hansen, T., Jensen, K., Pfeiffer, P., Alkhaldi, A., Brittenden, J., Jackson, A., Kamposioras, K., Kumaran, G., Macklin, C., Alexander, J., Harle, A., Hickish, T., Talbot, R., Tarver, D., Bridgewater, J., Partridge, W., Sundaresan, V., Vivekanandan, S., Agrawal, N., Higginson, A., Muthuramalingam, S., O’Leary, D., Devarajan, G., Gulati, M., Kerwat, R., Maisey, N., Mikhaeel, G., Ismail, T., Middleton, G., Page, A., Steven, N., Taniere, P., Gutmann, J., Huang, J., Raouf, S., Dunn, W., Escola, C. Lopez, Potter, V., Scholefield, J., Walker, G., Zaitoun, A., Eason, D., McPhail, N., Mmeka, W., Stenhouse, G., Watson, A., Fozard, B., Hickish, T., Snape, S., Ellis, R., Faux, W., Jenkins, R., Maskell, G., Kulkarni, R., Lund, J., Menon, S., Singh, R., Chandler, I., Daniels, I., Harries, S., Osborne, M., Bell, J., Krell, D., Mayer, A., Ogunbiyi, O., Watkins, J., Bronder, C., Eaton, D., Taylor, A., Brown, G., Cunningham, D., Tekkis, P., Wotherspoon, A., Dobson, M., Mitchell, P., Pitt, M., Scott, N., Susnerwala, S., Adab, F., Britton, I., Ghiridaran, S., Howitt, C., Kirby, R., Biddlestone, L., Dalton, S., De Winton, E., Phillips, A., Ferry, D., Grumett, S., Kawesha, A., Maleki, K., Momtahan, N., Burnett, H., Hayes, S., Soop, M., Branagan, G., Cook, I., Cook, S., Iveson, T., Shablak, A., Coup, A., Hamid, A., Moore, P., O’Toole, L., Pai, D., Bateman, A., Bateman, A., Blaquiere, R., Nichols, P., Chappell, M., Dworkin, M., Jain, S., Tsang, D., Hopkins, K., Loveday, E., Lyons, A., Rooney, N., Ali, N., Chatterjee, M., Chiphang, A., Dundas, S., Myint, A. Sun, Zeiderman, M., Beharry, N., Chong, H., Lofts, F., Melville, D., Finan, P., Seymour, M., Tolan, D., West, N., Anyamene, N., Burling, D., Kennedy, R., Moorghen, M., Agrawal, S., Hasan, J., Mehta, S., Saeed, M., Burgess, P., John, L., Lowndes, S., Planner, A., Campbell, F., Hughes, M., Rooney, P., Smith, D., Hochhauser, D., Obichere, A., Rodriguez-Justo, M., Shiu, K., Taylor, S., Correa, P., James, S., Shatwell, W., Williams, N., Brady, J., Lanaspre, E., Mikhaeel, G., Ahmad, M., Gill, T., Wilson, D., Adams, R., Beehen, R., Morgan, M., Lindh, B., Adams, R., Morgan, M., Ford, A., Gopal, K., Pranesh, N., Shareef, D., Tighe, M., Busby, K., Correa, P., Sanders, S., Sinha, R., Ahmad, R., Desai, S., Ramesh, S., Hilman, S., Lott, M., O’Brien, J., Radstone, D., West, D., Amin, S., Hampton, J., Hornbuckle, J., Kitsanta, P., Ali, M., Desai, A., Hadaki, M., Hall, M., Arul, D., Hochhauser, D., Leonard, P., Mukhtar, H., Murray, D., Baxter, A., Churn, M., Farrugia, D., Lake, S., Smith, G., Bansal, A., Chandran, P., Corr, C., Gollins, S., Davenport, A., Saunders, M., Sukumar, S., Bathurst, N., Beaumont, E., Cooper, E., Francis, N., Sephton, M., Sparrow, G., Clarke, A., Haselden, J., Last, K., Woodcock, N., Atkinson, M., Gollins, S., Gupta, M., Maw, A., Abdullah, N., Bale, C., and Lord, M.

Published
2023
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6. Individual identification of flapper skate Dipturus intermedius using a baited camera lander

Author

Benjamins, S, Fox, CJ, Last, K, and McCarty, CE

Subjects
Zoology, QL1-991, Botany, QK1-989
Abstract

The ability of baited remote underwater video (BRUV) landers to attract and identify individual flapper skates Dipturus intermedius based on their spot patterns was tested within the Loch Sunart to Sound of Jura Marine Protected Area (MPA). Two BRUV lander designs, baited with a mix of mackerel, herring, squid and octopus, were deployed in 9 different locations at depths between 105 and 133 m for approx. 2 h per deployment. The BRUV successfully obtained video footage and still images of flapper skates during 4 deployments. On 6 occasions, skates remained at the bait long enough for us to obtain high-quality video of their dorsal spot patterns. Five individuals, one of which was observed at 2 adjacent landers in quick succession, were all uniquely distinguishable; one female was matched to existing records in a photo-identification catalogue based on images collected by recreational sea anglers within the MPA. BRUVs offer significant potential for studying individual presence, distribution patterns and abundance of flapper skates, independent of fishing/angling efforts. BRUV landers can therefore support monitoring efforts for evaluating long-term conservation management measures for this species.

Published
2018
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17. A randomised phase II trial and feasibility study of palliative chemotherapy in frail or elderly patients with advanced gastroesophageal cancer (321GO)

Author

Hall, PS, Lord, SR, Collinson, M, Marshall, H, Jones, M, Lowe, C, Howard, H, Swinson, D, Velikova, G, Anthoney, A, Roy, R, Dent, J, Cheeseman, S, Last, K, and Seymour, MT

Subjects
Male, oesophageal cancer, Cancer Research, medicine.medical_specialty, Palliative care, Esophageal Neoplasms, Organoplatinum Compounds, Frail Elderly, Population, Adenocarcinoma, Deoxycytidine, Capecitabine, 03 medical and health sciences, 0302 clinical medicine, Internal medicine, Antineoplastic Combined Chemotherapy Protocols, medicine, Clinical endpoint, Humans, 030212 general & internal medicine, education, Aged, Epirubicin, Aged, 80 and over, education.field_of_study, business.industry, gastric cancer, Standard treatment, Palliative Care, feasibility study, clinical trial, ageing and cancer, Surgery, Oxaliplatin, Clinical trial, Regimen, Oncology, 030220 oncology & carcinogenesis, Disease Progression, Clinical Study, Feasibility Studies, Female, Fluorouracil, business, medicine.drug
Abstract

BACKGROUND: Elderly patients are commonly under-represented in cancer clinical trials. The 321GO was undertaken in preparation for a definitive phase three trial assessing different chemotherapy regimens in a frail and/or elderly population with advanced gastroesophageal (GO) cancer. METHODS: Patients with advanced GO cancer considered unfit for conventional dose chemotherapy were randomly assigned in a 1 : 1 : 1 ratio to: epirubicin, oxaliplatin and capecitabine (EOX); oxaliplatin and capecitabine (OX); and capecitabine alone (X) (all 80% of full dose and unblinded). The primary end point was patient recruitment over an 18-month period. A registration study recorded treatment choice for all patients with advanced GO cancer at trial centres. RESULTS: A total of 313 patients were considered for palliative chemotherapy for GO cancer over the 18-month period: 115 received full dose treatment, 89 less than standard treatment or entered 321GO and 111 no treatment. Within 321GO, 55 patients were randomly assigned (19 to OX and X; 17 to EOX). Progression-free survival (PFS) for all patients was 4.4 months and by arm 5.4, 5.6 and 3.0 months for EOX, OX and X, respectively. The number of patients with a good overall treatment utility (OTU), a novel patient-centred endpoint, at 12 weeks was 3 (18%), 6 (32%) and 1 (6%) for EOX, OX and X, respectively. At 6 weeks, 22 patients (41%) had experienced a non-haematologic toxicity ⩾grade 3, most commonly lethargy or diarrhoea. The OTU was prognostic for overall survival in patients alive at week 12 (logrank test P=0.0001). CONCLUSIONS: It is feasible to recruit elderly and/or frail patients with advanced GO cancer to a randomised clinical trial. The OX is the preferred regimen for further study. Overall treatment utility shows promise as a comparator between treatment regimens for feasibility and randomised trials in the elderly and/or frail GO cancer population.

Published
2017

19. Monitoring of Tübingen Endosseous Dental Implants by Glycosaminoglycans Analysis of Gingival Crevicular Fluid.

Author

Last, K. S., Cawood, J. I., Howell, R. A., and Embery, G.

Subjects
ENDOSSEOUS dental implants, GLYCOSAMINOGLYCANS, DENTAL implants, FLUIDS, ARTIFICIAL implants, ORAL surgery
Abstract

Glycosaminoglycans (GAG) in gingival crevicular fluid (GCF) samples were determined by cellulose acetate electrophoresis and densitometric scanning. Two GAG bands, hyaluronic acid and chondroitin-4-sulphate (C4S), were detected in GCF from implants, similar to the profile from teeth. High GCF volumes and GAG contents, notably C4S, may reflect postoperative alveolar bone responses, particularly resorption, at different stages of healing and function of successful implants. They may also indicate adverse tissue changes in failing implants. A comparison of crowned implants and matched teeth suggests that the periodontal ligament contributes to the GCF GAG profile. This may be a useful laboratory method of monitoring implants to detect adverse tissue responses at an early stage. [ABSTRACT FROM AUTHOR]

Published
1991

21. Aggrecan is degraded by matrix metalloproteinases in human arthritis. Evidence that matrix metalloproteinase and aggrecanase activities can be independent

Author

Amanda Fosang, Last, K., and Maciewicz, R. A.

Subjects
Male, Proteolysis, Immunoblotting, Population, Arthritis, Matrix metalloproteinase, Epitopes, chemistry.chemical_compound, Endopeptidases, Synovial Fluid, Centrifugation, Density Gradient, medicine, Humans, Synovial fluid, Lectins, C-Type, Aggrecans, Chondroitin sulfate, education, Aggrecan, Aggrecanase, Extracellular Matrix Proteins, education.field_of_study, medicine.diagnostic_test, Antibodies, Monoclonal, Metalloendopeptidases, General Medicine, Chromatography, Agarose, medicine.disease, Molecular biology, Peptide Fragments, chemistry, Biochemistry, Female, Proteoglycans, Research Article
Abstract

Proteolytic degradation of aggrecan is a hallmark of the pathology of arthritis, yet the identity of the enzyme(s) in cartilage responsible for this degradation is unknown. Previous studies have suggested that the matrix metalloproteinases (MMPs) may be involved but there has been no definitive evidence for their direct action in the proteolysis of aggrecan in human arthritis. We now show unequivocally that aggrecan fragments derived from the specific action of MMPs can be detected in synovial fluids from patients with both inflammatory and noninflammatory arthritis, with a neoepitope monoclonal antibody AF-28 that detects the NH2-terminal sequence F342FGVG.... The synovial fluid MMP fragments were of low buoyant density and distributed exclusively at the top of cesium chloride density gradients, suggesting that these fragments lacked chondroitin sulfate chains. AF-28 immunoblotting of synovial fluid aggrecan fragments revealed a population of small AF-28 fragments of 30-50 kD. Based on their size relative to characterized products of an MMP-8 digest (Fosang, A.J., K. Last, P. Gardiner, D.C. Jackson, and L. Brown. 1995, Biochem. J. 310:337-343), these AF-28 fragments were derived from proteinase cleavage at, or near, the ...ITEGE373 / ARGSV... aggrecanase site. Immunodetection with polyclonal anti-ITEGE antiserum revealed that these fragments lacked the ...ITEGE374 COOH terminus and were not therefore products of aggrecanase action. The same fluid samples contained a broad 68-90-kD G1 fragment that contained the COOH-terminal ...ITEGE374 neoepitope. The results suggest that in some circumstances, despite extensive proteolysis of the core protein, aggrecan molecules may be cleaved by MMPs or aggrecanase in the interglobular domain, but not both.

Published
1996

22. Calanus finmarchicusseasonal cycle and diapause in relation to gene expression, physiology, and endogenous clocks

Author

Häfker, N. Sören, Teschke, M., Last, K. S., Pond, D. W., Hüppe, L., and Meyer, B.

Abstract

The copepod Calanus finmarchicusplays a crucial role in the north Atlantic food web. Its seasonal life cycle involves reproduction and development in surface waters before overwintering in diapause at depth. Although diapause has been studied for more than a century, the factors responsible for the initiation and termination of it are still unclear. Endogenous clocks have been identified as potent tools for photoperiod measurement and seasonal rhythmicity in many terrestrial species, but knowledge of these remains scarce in the marine realm. Focusing on the dominant CV copepodid stage, we sampled a population of C. finmarchicusfrom a Scottish sea loch to characterize population dynamics, several physiological parameters, and diel and seasonal expression rhythms of 35 genes representing different metabolic pathways, including the circadian clock machinery. This generated a detailed overview of the seasonal cycle of C. finmarchicusincluding the most extensive field dataset on circadian clock gene expression in a marine species to date. Gene expression patterns revealed distinct gene clusters upregulated at different phases of the copepod's seasonal cycle. While diel clock cycling was restricted to the active spring/summer phase, many clock genes exhibited the highest expression during diapause. Our results provide new insights into diapause on physiological and genetic levels. We suggest that photoperiod, in interaction with internal and external factors (lipid content, temperature, food availability) and the endogenous clock mechanism, plays an important role in the timing of diapause in C. finmarchicus.

Published
2018
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27. Neutrophil collagenase (MMP-8) cleaves at the aggrecanase site E 373 –A 374 in the interglobular domain of cartilage aggrecan

Author

Hughes, C E, Fosang, A J, Last, K, Murphy, G, Hardingham, T E, Knäuper, V, Tschesche, H, Caterson, B, and Neame, P J

Abstract

Native and recombinant neutrophil collagenase (MMP-8) was shown to cleave at the E(373)-A(374) 'aggrecanase'' site in the interglobular domain of aggrecan. The time course of digestion in vitro showed that MMP-8 cleaved initially at N-341-F-342, the predominant metalloproteinase site, before cleaving at the E(373)-A(374) site. A synthetic peptide, IPENFFG, inhibited cleavage at E(373)-A(374) but not N-341-F-342 in vitro, indicating that the E(373)-A(374) sequence was a less preferred site for MMP-8 cleavage than N-341-F-342. IPENFFG also inhibited release of A(374) RGSVI fragments from cartilage in explant culture, suggesting that a metalloproteinase cleaved at the aggrecanase site in situ. The possibility remains that 'aggrecanase' may be a metalloproteinase in cartilage.

Published
1994
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29. FIBROBLAST AND NEUTROPHIL COLLAGENASES CLEAVE AT 2 SITES IN THE CARTILAGE AGGRECAN INTERGLOBULAR DOMAIN

Author

FOSANG, AJ, LAST, K, KNAUPER, V, NEAME, PJ, MURPHY, G, HARDINGHAM, TE, Tschesche, Harald, and HAMILTON, JA

Subjects
musculoskeletal system
Abstract

The actions of recombinant human fibroblast collagenase (MMP1), purified polymorphonuclear leucocyte collagenase (MMP8) and their N-terminal catalytic domain fragments against cartilage aggrecan and an aggrecan GI-G2 fragment have been investigated in vitro. After activation with recombinant human stromelysin and trypsin, both collagenases were able to degrade human and porcine aggrecans to a similar extent. An N-terminal G1-G2 fragment (150 kDa) was used to identify specific cleavage sites occurring within the proteinase-sensitive interglobular domain between G1 and G2. Two specific sites were found; one at an Asn341-Phe342 bond and another at Asp441-Leu442 (human sequence). This specificity of the collagenases for aggrecan G1-G2 was identical with that of the truncated metalloproteinase matrilysin (MMP7), but different from those of stromelysin (MMP3) and the gelatinases (MMP2 or gelatinase A; MMP9 or gelatinase B) which cleave at the Asn-Phe site, but not the Asp-Leu site. In addition, collagenase catalytic fragments lacking C-terminal hemopexin-like domains were tested and shown to exhibit the same specificities for the G1-G2 fragment as the full-length enzymes. Thus the specificity of the collagenases for cartilage aggrecan was not influenced by the presence or absence of the C-terminal domain. Together with our previous findings, the results show that stromelysin-1, matrilysin, gelatinases A and B and fibroblast and neutrophil collagenases cleave at a common, preferred site in the aggrecan interglobular domain, and additionally that both fibroblast and neutrophil collagenases cleave at a second site in the interglobular domain that is not available to stromelysin or gelatinases.

Published
1993

44. Sexual Satellites, Moonlight and the Nuptial Dances of Worms: the Influence of the Moon on the Reproduction of Marine Animals.

Author

Bentley, M., Olive, P., and Last, K.

Abstract

The evidence that the moon has a profound effect on the timing of reproductive activities of marine animals is compelling. Some moon phase related spawning events are revealed by the constant phase relationship between the timing of “once per year” spawning events and the lunar phase as in the highly synchronised breeding of the palolo worm Palola viridis and the Japanese crinoid Comanthus japonicus In other cases there is a repeated lunar cycle of reproductive activity and again the marine worms provide many good examples. The breeding of the palolo worm involves the highly synchronised release of what are in effect detached sexual satellites and the timing of this has annual (solar year), lunar, daily and tidal rhythm components. In a similar way, the onset of sexual maturation and participation in the nuptial dance of Platynereis dumerilli has strong lunar components. Sexual reproduction is the culmination of a process of sexual maturation that takes many months for completion and the mechanisms by which moon phase relationships are imposed on this process must have been selected for by mechanisms relating to reproductive success. The polychaetes provide excellent models for investigation of both the selective advantage and the physiological processes involved in reproductive synchrony. We have recently shown that the spawning of the lugworm Arenicola marina has lunar components and we conclude that an interaction between solar and lunar signals is widespread in the timing of reproduction in marine animals. Carl Hauenschild was the first to demonstrate the existence of a free-running circa-lunar rhythm in marine animals using captive populations of Platynereis dumerilli His experiments also provided clear evidence for the influence of moonlight (light at night) as the zeitgeber for this rhythm. This implies a high level of sensitivity to light, and the operation of appropriate endogenous biological rhythms. Using Nereis virens we have demonstrated a high level of sensitivity to low intensity solar light signals in relation to rhythmic processes with the properties similar to the circadian and photoperiodic mechanisms of terrestrial organisms. The spawning of N. virens is also believed to have lunar components reflecting the complex influences of the sun and the moon in the marine domain, where influences of the moon and sun are more equal. We suggest that evolution of biological rhythmicity that can be entrained to either lunar or solar systems arose in the marine domain. Recent advances in understanding the molecular components of the circadian clock system suggest that a search for a common molecular mechanism for both lunar and solar related biological rhythms in marine organisms might be very fruitful. [ABSTRACT FROM AUTHOR]

Published
1998
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46. Being a parent at ECCMID 2019.

Author

Last, K. and Papan, C.

Subjects
*PARENTS, *SCIENTIFIC community, *DIAPERS, *DIAGNOSTIC microbiology
Abstract

Rebecca Calisi and the Working Group of Mothers in Science have published a highly cited paper last year in which they address the childcare-conference conundrum [5]. 4 The Lancet Infectious Diseases, Gender parity in infectious diseases, Lancet Infect Dis, 19, 2019, 217The Lancet Infectious Diseases. 5 R.M. Calisi, Opinion: how to tackle the childcare-conference conundrum, Proc Natl Acad Sci USA, 115, 2018, 2845-2849Calisi, R.M. Opinion: How to tackle the childcare-conference conundrum. [Extracted from the article]

Published
2019
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47. Mutations in the interglobular domain of aggrecan alter matrix metalloproteinase and aggrecanase cleavage patterns. Evidence that matrix metalloproteinase cleavage interferes with aggrecanase activity.

Author

Mercuri, F A, Maciewicz, R A, Tart, J, Last, K, and Fosang, A J

Abstract

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.

Published
2000

49. Recombinant human aggrecan G1-G2 exhibits native binding properties and substrate specificity for matrix metalloproteinases and aggrecanase.

Author

Mercuri, F A, Doege, K J, Arner, E C, Pratta, M A, Last, K, and Fosang, A J

Abstract

A recombinant human aggrecan G1-G2 fragment comprising amino acids Val(1)-Arg(656) has been expressed in Sf21 cells using a baculovirus expression system. The recombinant G1-G2 (rG1-G2) was purified to homogeneity by hyaluronan-Sepharose affinity chromatography followed by high performance liquid chromatography gel filtration, and gave a single band of M(r) 90,000-95,000 by silver stain or immunoblotting with monoclonal antibody 1-C-6. The expressed G1-G2 bound to both hyaluronan and link protein indicating that the immunoglobulin-fold motif and proteoglycan tandem repeat loops of the G1 domain were correctly folded. Further analysis of secondary structure by rotary shadowing electron microscopy confirmed a double globe appearance, but revealed that the rG1-G2 was more compact than its native counterpart. The size of rG1-G2 by SDS-polyacrylamide gel electorphoresis was unchanged following digestion with keratanase and keratanase II and reduced by only 2-5 kDa following digestion with either O-glycosidase or N-glycosidase F. Recombinant G1-G2 was digested with purified matrix metalloproteinases (MMP), isolated aggrecanase, purified atrolysin C, or proteinases present in conditioned medium from cartilage explant cultures, and the products analyzed on SDS gels by silver stain and immunoblotting. Neoepitope antibodies recognizing the N-terminal F(342)FGVG or C-terminal DIPEN(341) sequences were used to confirm MMP cleavage at the Asn(341) downward arrow Phe bond, while neoepitope antibodies recognizing the N-terminal A(374)RGSV or C-terminal ITEGE(373) sequences were used to confirm aggrecanase cleavage at the Glu(373) downward arrow Ala bond. Cleavage at the authentic MMP and aggrecanase sites revealed that these proteinases have the same specificity for rG1-G2 as for native aggrecan. Incubation of rG1-G2 with conditioned medium from porcine cartilage cultures revealed that active soluble aggrecanase but no active MMPs, was released following stimulation with interleukin-1alpha or retinoic acid. Atrolysin C, which cleaves native bovine aggrecan at both the aggrecanase and MMP sites, efficiently cleaved rG1-G2 at the aggrecanase site but failed to cleave at the MMP site. In contrast, native glycosylated G1-G2 with or without keratanase treatment was cleaved by atrolysin C at both the aggrecanase and MMP sites. The results suggest that the presence or absence per se of keratan sulfate on native G1-G2 does not affect the activity of atrolysin C toward the two sites.

Published
1999

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