We demonstrate that in humans, two metalloproteases, ADAMTS-9 (1935 amino acids) and ADAMTS-20 (1911 amino acids) are orthologs of GON-1, an ADAMTS protease required for gonadal morphogenesis in Caenorhabditis elegans. ADAMTS-9 and ADAMTS-20 have an identical modular structure, are distinct in possessing 15 TSRs and a unique C-terminal domain, and have a similar gene structure, suggesting that they comprise a new subfamily of human ADAMTS proteases. AD-AMTS20 is very sparingly expressed, although it is detectable in epithelial cells of the breast and lung. However, ADAMTS9 is highly expressed in embryonic and adult tissues, and therefore we characterized the AD-AMTS-9 protein further. Although the ADAMTS-9 zymogen has many proprotein convertase processing sites, pulse-chase analysis, site-directed mutagenesis, and amino acid sequencing demonstrated that maturation to the active form occurs by selective proprotein convertase (e.g. furin) cleavage of the Arg 287 -Phe 288 bond. Although lacking a transmembrane sequence, ADAMTS-9 is retained near the cell surface as well as in the ECM of transiently transfected COS-1 and 293 cells. COS-1 cells transfected with ADAMTS9 (but not vector-transfected cells) proteolytically cleaved bovine versican and aggrecan core proteins at the Glu 441 -Ala 442 bond of versican V1 and the Glu 1771 -Ala 1772 bond of aggrecan, respectively. In contrast, the ADAMTS-9 catalytic domain alone was neither localized to the cell surface nor able to confer these proteolytic activities on cells, demonstrating that the ancillary domains of ADAMTS-9, including the TSRs, are required both for specific extracellular localization and for its versicanase and aggrecanase activities.

OBJECTIVETo minimize hypoglycemia in subjects with type 1 diabetes by automated glucagon delivery in a closed-loop insulin delivery system.RESEARCH DESIGN AND METHODSAdult subjects with type 1 diabetes underwent one closed-loop study with insulin plus placebo and one study with insulin plus glucagon, given at times of impending hypoglycemia. Seven subjects received glucagon using high-gain parameters, and six subjects received glucagon in a more prolonged manner using low-gain parameters. Blood glucose levels were measured every 10 min and insulin and glucagon infusions were adjusted every 5 min. All subjects received a portion of their usual premeal insulin after meal announcement.RESULTSAutomated glucagon plus insulin delivery, compared with placebo plus insulin, significantly reduced time spent in the hypoglycemic range (15 ± 6 vs. 40 ± 10 min/day, P = 0.04). Compared with placebo, high-gain glucagon delivery reduced the frequency of hypoglycemic events (1.0 ± 0.6 vs. 2.1 ± 0.6 events/day, P = 0.01) and the need for carbohydrate treatment (1.4 ± 0.8 vs. 4.0 ± 1.4 treatments/day, P = 0.01). Glucagon given with low-gain parameters did not significantly reduce hypoglycemic event frequency (P = NS) but did reduce frequency of carbohydrate treatment (P = 0.05).CONCLUSIONSDuring closed-loop treatment in subjects with type 1 diabetes, high-gain pulses of glucagon decreased the frequency of hypoglycemia. Larger and longer-term studies will be required to assess the effect of ongoing glucagon treatment on overall glycemic control.

The amino and carboxyl propeptides of procollagens I and II are removed by specific enzymes as a prerequisite for fibril assembly. Null mutations in procollagen I Npropeptidase (ADAMTS-2) cause dermatosparaxis in cattle and the Ehlers-Danlos syndrome (dermatosparactic type) in humans by preventing proteolytic excision of the N-propeptide of procollagen I. We have found that procollagen II is processed normally in dermatosparactic nasal cartilage, suggesting the existence of another N-propeptidase(s). We investigated such a role for ADAMTS-3 in Swarm rat chondrosarcoma RCS-LTC cells, which fail to process the procollagen II N-propeptide. Stable transfection of RCS-LTC cells with bovine ADAMTS-2 or human ADAMTS-3 partially rescued the processing defect, suggesting that ADAMTS-3 has procollagen II N-propeptidase activity. Human skin and skin fibroblasts showed 30-fold higher mRNA levels of ADAMTS-2 than AD-AMTS-3, whereas ADAMTS-3 mRNA was 5-fold higher than ADAMTS-2 mRNA in human cartilage. We propose that both ADAMTS-2 and ADAMTS-3 process procollagen II, but ADAMTS-3 is physiologically more relevant, given its preferred expression in cartilage. The findings provide an explanation for the sparing of cartilage in dermatosparaxis and, perhaps, for the relative sparing of some procollagen I-containing tissues.Collagens consist of the major structural proteins of the extracellular matrix (ECM) 1 and exist in both fibril-forming (e.g. collagens I-III, V, and XI) and nonfibrillar forms (1, 2). Molecules belonging to both categories are homotrimeric (e.g. collagen II) or heterotrimeric (e.g. collagen I) assemblies of specific ␣ chains, each the product of a single gene (1, 2). The molecular types of collagen, as well as the specific supramolecular aggregates they form, are often tissue-specific and provide a specialized function. For example, collagen I, the principal collagen of skin, is arranged in randomly oriented bundles in the dermis but in parallel bundles in tendons. Collagen II, a specific component of cartilage ECM, is arranged in an open meshwork that traps proteoglycans and facilitates resistance to compression. The synthesis, secretion, and assembly of collagens into specific supramolecular aggregates is a complex, multistep process (3, 4). Fibrillar collagens I-III are synthesized as a soluble procollagen monomer comprising a long triple helical "collagenous" region with smaller polypeptide extensions (propeptides) at the amino and carboxyl ends (4). Removal of the propeptides by specific enzymes, the N-and C-propeptidases (proteinases), is a prerequisite for the correct assembly of collagens I and II into growing fibrils (3, 4). The procollagen C-propeptidase is identical to bone morphogenetic protein-1 and processes all three of these fibrillar collagens (5). Biochemically distinct N-propeptidases with specificity for procollagens I and II or procollagen III are known (6). The bovine and human procollagen I N-propeptidases have been cloned (7,8). This enzyme (designated ADAMTS-2, EC 3.4.24.14), is a zinc...

Glucagon may fail to prevent hypoglycemia when insulin on board is high or when glucagon delivery is delayed due to overestimation of glucose by the sensor. Improvements in sensor accuracy and delivery of larger or earlier glucagon doses when insulin on board is high may further reduce the frequency of hypoglycemia.

Under certain conditions, aqueous solutions of glucagon and MAR-D28 are stable for at least 5 days and are thus very likely to be safe in mammals. Glycine buffer at a pH of 10 appears to be optimal for avoiding cytotoxicity and amyloid fibril formation.