In this work, we characterized 2 novel insecticidal proteins; Vip3Ab1 and Vip3Bc1. These proteins display unique insecticidal spectra and have differential rates of processing by lepidopteran digestive enzymes. Furthermore, we have found that both proteins exist as tetramers in their native state before and after proteolysis. In addition, we expressed truncated forms and protein chimeras to gain a deeper understanding of toxin specificity and stability. Our study confirms a role for the C-terminal 65 kDa domain in directing insect specificity. Importantly, these data also indicate a specific interaction between the 20 kDa amino terminus and 65 kDa carboxy terminus, after proteolytic processing. We demonstrate the C-terminal 65 kDa to be labile in native proteolytic conditions in absence of the 20 kDa N-terminus. Thus, the 20 kDa fragment functions to provide stability to the C-terminal domain, which is necessary for lethal toxicity against lepidopteran insects.

Insect pests are a major cause of crop losses worldwide, with an estimated economic cost of $470 billion annually. Biotechnological tools have been introduced to control such insects without the need for chemical pesticides; for instance, the development of transgenic plants harbouring genes encoding insecticidal proteins. The Vip3 (vegetative insecticidal protein 3) family proteins from Bacillus thuringiensis convey toxicity to species within the Lepidoptera, and have wide potential applications in commercial agriculture. Vip3 proteins are proposed to exert their insecticidal activity through pore formation, though to date there is no mechanistic description of how this occurs on the membrane. Here we present cryo-EM structures of a Vip3 family toxin in both inactive and activated forms in conjunction with structural and functional data on toxin–membrane interactions. Together these data demonstrate that activated Vip3Bc1 complex is able to insert into membranes in a highly efficient manner, indicating that receptor binding is the likely driver of Vip3 specificity.

ADAMTS-4 and ADAMTS-Aggrecanase-mediated degradation of aggrecan, the major aggregating proteoglycan of articular cartilage, is an early and sustained feature of osteoarthritis (OA (3, 4). Because of their preference for Glu at P1, both ADAMTS-4 and -5 are considered glutamyl endoproteinases. Whereas ADAMTS-5 is constitutively expressed in human cartilage, ADAMTS-4 is inducible by a number of inflammatory cytokines, such as interleukin-1␤ and tumor necrosis factor-␣ (5). Gene knockout of ADAMTS-5, but not ADAMTS-4, expression in mice has been shown to be chondroprotective in a surgical mouse model of OA (6, 7), yet in human OA cartilage explants both ADAMTS-4 and ADAMTS-5 mediate aggrecan breakdown (8). Inhibition of ADAMTS-4 and ADAMTS-5 activity may represent a viable option for slowing down the progression of cartilage deterioration in OA.Alignment of the known sequences flanking the ADAMTS-4 cleavage sites in the proteoglycan substrates, aggrecan, versican, and brevican, led to the proposal of a 24-amino acid consensus motif (9). Not surprisingly, a glutamic acid residue occupied P1 (100% conserved) with P2Ј occupied by the basic amino acids, Arg or Lys. The authors speculated that activity of ADAMTS family members toward proteoglycan substrates was primarily dictated by an extended 23-amino acid motif N-terminal to the scissile bond, and a short 3-amino acid motif downstream of the site of cleavage. However, unlike the scissile bonds in the aggregating proteoglycans, the site of ADAMTS-4 proteolysis in ␣ 2 -macroglobulin (␣ 2 M) is Met 690 /Gly 691 , with no requirement for Glu at P1 (10). Yet, P1Ј to P3Ј in ␣ 2 M, Gly-Arg-Gly, is remarkably similar to downstream sequences in aggrecan and brevican, implying that PЈ amino acids may be more important in recognition and catalysis than sequences upstream of the scissile bond. ADAMTS-4 has also been shown * The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Objective. Fibronectin fragments are present at high concentrations in the cartilage of patients with rheumatoid arthritis and patients with osteoarthritis (OA) and have been shown to promote cartilage catabolism in human cartilage cultures, suggesting that fibronectin fragments participate in the initiation and progression of arthritic disease. This study was undertaken to 1) identify the major fibronectin fragments in human OA cartilage and confirm their ability to elicit cartilage catabolism, 2) identify the cleavage sites in fibronectin and generate the corresponding neoepitope antibodies, and 3) explore the utility of fibronectin neoepitopes as biomarkers.Methods. Fibronectin fragments were purified from human OA cartilage using affinity chromatography; their N-termini were then identified by sequencing. Bovine nasal cartilage was treated with affinity-purified fibronectin fragments and assayed for aggrecan breakdown by monitoring the release of glycosaminoglycans and the aggrecan neoepitope 1771 AGEG. Fibronectin neoepitopes were detected by Western blotting in cytokine-treated media of human cartilage explants, and by immunohistochemical analyses of human OA cartilage.Results. Multiple fibronectin fragments were isolated from human OA cartilage, and all contained the N-terminus 272 VYQP. These fragments induced aggrecanase-mediated cartilage catabolism in bovine cartilage explants. Fibronectin fragments with the N-terminus 272 VYQP and fragments with the C-terminus VRAA 271 were detected following cytokine treatment of human cartilage extracts. These neoepitopes localized with areas of aggrecan loss in OA cartilage.Conclusion. Human OA cartilage contains fibronectin fragments with catabolic activity and a major cleavage site within fibronectin. This study is the first to characterize fibronectin neoepitopes in OA cartilage, suggesting that they may represent a novel biomarker of arthritis.

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