Identification of a Novel HtrA1-susceptible Cleavage Site in Human Aggrecan: EVIDENCE FOR THE INVOLVEMENT OF HtrA1 IN AGGRECAN PROTEOLYSIS IN VIVO

Mass spectrometry-based proteomic analyses performed on cartilage tissue extracts identified the serine protease HtrA1/PRSS11 as a major protein component of human articular cartilage, with elevated levels occurring in association with osteoarthritis. Overexpression of a catalytically active form of HtrA1, but not an active site mutant (S328A), caused a marked reduction in proteoglycan content in chondrocyte-seeded alginate cultures. Aggrecan degradation fragments were detected in conditioned media from the alginate cultures overexpressing active HtrA1. Incubation of native or recombinant aggrecan with wild type HtrA1 resulted in distinct cleavage of these substrates. Cleavage of aggrecan by HtrA1 was strongly enhanced by HtrA1 agonists such as CPII, a C-terminal hexapeptide derived from the C-propeptide of procollagen IIα1 (i.e. chondrocalcin). A novel HtrA1-susceptible cleavage site within the interglobular domain (IGD) of aggrecan was identified, and an antibody that specifically recognizes the neoepitope sequence (VQTV³⁵⁶) generated at the HtrA1 cleavage site was developed. Western blot analysis demonstrated that HtrA1-generated aggrecan fragments containing the VQTV³⁵⁶ neoepitope were significantly more abundant in osteoarthritic cartilage compared with cartilage from healthy joints, implicating HtrA1 as a critical protease involved in proteoglycan turnover and cartilage degradation during degenerative joint disease.The mammalian high-temperature requirement A (HtrA) family of serine proteases is defined by a characteristic trypsin-like serine protease domain and one or two C-terminal PDZ domains. Four mammalian HtrA proteins have been identified to date, HtrA1–4. HtrA1 (also called PRSS11) is a ubiquitously expressed extracellular serine protease which contains a signal sequence for secretion, an insulin-like growth factor (IGF)²-binding protein domain, and a Kazal-type serine protease inhibitor domain in addition to the serine protease domain and one C-terminal PDZ domain (1). HtrA1 has been implicated in the progression of several pathologies including age-related macular degeneration, cancer, Alzheimer disease, rheumatoid arthritis, and osteoarthritis (OA) (2–10). HtrA1 has also been shown to inhibit osteoblast mineralization (11).Expression of HtrA1 has been found to be elevated in articular cartilage in association with OA (5). In addition, HtrA1 levels are up-regulated in murine cartilage after experimentally induced joint damage (6). The physiological role of HtrA1 in OA disease progression as well as in other pathologies is unclear. Preliminary studies using in vitro digestion assays suggest that HtrA1 might be capable of digesting cartilage extracellular matrix (ECM) proteins such as fibromodulin, cartilage oligomeric matrix protein (COMP), fibronectin, decorin, and aggrecan (6, 12, 13). Furthermore, it was recently reported that elevated levels of HtrA1 protein (∼7-fold above normal) are present in synovial fluids obtained from OA patients and that fibronectin fragments generated by HtrA1 cleavage induced the expression of catabolic enzymes such as matrix metalloproteinases-1 (MMP-1) and MMP-3 in synovial fibroblasts (4). HtrA1 has also been shown to modulate multiple signaling pathways in vitro. It binds to transforming growth factor-β family proteins including transforming growth factor-β1 and bone morphogenetic proteins 2 and 4 and inhibits signaling mediated by these factors (14, 15). In addition, HtrA1 has been shown to cleave IGF-binding protein-5 and possibly regulate signaling mediated by IGF (16). These findings suggest that the protease HtrA1 may play a physiological role in cartilage during OA.Articular cartilage is made up of chondrocytes surrounded by the ECM comprised mainly of the proteoglycan, aggrecan, and type II collagen. During normal homeostasis there is a dynamic balance between anabolic activities such as proteoglycan synthesis as well as catabolic activities in which the ECM is destroyed. When the catabolic activities of proteases, such as MMPs and aggrecanases, offset new matrix synthesis, focal degradation and loss of articular cartilage occurs, resulting in the development of OA. In some in vitro digestion studies, we and others have shown degradation of aggrecan by recombinant HtrA1 (6, 12, 13). In the present study we set out to examine the physiological relevance of aggrecan cleavage by HtrA1 in OA disease progression.     

Computational and analytical framework for small RNA profiling by high-throughput sequencing

The advent of high-throughput sequencing (HTS) methods has enabled direct approaches to quantitatively profile small RNA populations. However, these methods have been limited by several factors, including representational artifacts and lack of established statistical methods of analysis. Furthermore, massive HTS data sets present new problems related to data processing and mapping to a reference genome. Here, we show that cluster-based sequencing-by-synthesis technology is highly reproducible as a quantitative profiling tool for several classes of small RNA from Arabidopsis thaliana. We introduce the use of synthetic RNA oligoribonucleotide standards to facilitate objective normalization between HTS data sets, and adapt microarray-type methods for statistical analysis of multiple samples. These methods were tested successfully using mutants with small RNA biogenesis (miRNA-defective dcl1 mutant and siRNA-defective dcl2 dcl3 dcl4 triple mutant) or effector protein (ago1 mutant) deficiencies. Computational methods were also developed to rapidly and accurately parse, quantify, and map small RNA data.     

Synthetic substrates as amine donors and acceptors in microbial transglutaminase-catalysed reactions

Microbial transglutaminase (EC 2.3.2.13) (mTGase) catalyses a calcium-independent acyl-transfer reaction in which -(γ-glutamyl)lysine bonds are formed using the γ-carboxyamide groups of peptide-bound glutamine residues and the amino group of lysine side-chains. Here we present a comparative study on alternative lysine and glutamine substitutes in mTGase catalysis. A homologous series of ω-amino acids, serving as lysine substitutes, was incorporated into carbobenzoxy-l-glutaminylglycine (CBZ-Gln-Gly). The rate constants and particular conversion rates increased with increasing chain length. As for the glutamine substitutes, adipic diamide, glutaric monoamide, and glutaric diamide were converted with monodansylcadaverine (DNS-cadaverine) under mTGase catalysis. For the synthetic glutamine substitutes, the substrates of natural chain length, glutaric mono- and diamide, are better converted than the longer adipic diamide indicating that the window of opportunity seems to be smaller. Synthetic substrates, serving as amine acceptors, offer new opportunities in the field of transglutaminase-catalysed reactions.     

Inconsistent estimates of diversity between traditional and DNA taxonomy in bdelloid rotifers

Microscopic animals offer great potential in the analysis of spatial patterns of diversity, as they may provide different scenarios for biogeography and macroecology, but understanding diversity of microscopic animals is hampered by lack of comprehensive data on species distribution and by unreliable taxonomy. DNA taxonomy may prove useful in obtaining reliable data in the future, but we still do not know to what extent traditional and DNA taxonomy can be comparable for microscopic organisms. In this paper, we compare analyses and estimates of diversity at the level of species assemblage between traditional and DNA taxonomy for a group of moss-dwelling microscopic animals, bdelloid rotifers. The results are straightforward: Traditional species identification underestimates diversity by factors of 2 at the local and 2.5 at the regional scale. We discuss the results in the framework of current hypotheses on the distribution of microscopic animals.     

The A1555G mitochondrial DNA mutation in Greek patients with non-syndromic, sensorineural hearing loss

Mitochondrial DNA mutations are undoubtedly a factor that contributes to sensorineural, non-syndromic deafness. One specific mutation, the A1555G, is associated with both aminoglycoside-induced and non-syndromic hearing impairment. The mutation is considered to be the most common of all mitochondrial DNA deafness-causing mutations but its frequency varies between different populations. Here we report on the first large screening of the A1555G mitochondrial DNA mutation in the Greek population. The aim of this study was to determine the frequency of the A1555G mutation in Greek sensorineural, non-syndromic deafness patients, with childhood onset. We screened 478 unrelated Greek patients with hearing loss of any degree and found two individuals harboring the A1555G mutation (0.42%). Both cases had been subjected to aminoglycosides. They were prelingual, familial and homoplasmic for the A1555G mutation. One of the cases was also found heterozygous for the frequent GJB2 35delG mutation, while the other case was negative. The A1555G mutation seems to be less common than in other European populations.