Cathepsin D cleaves aggrecan at unique sites within the interglobular domain and chondroitin sulfate attachment regions that are also cleaved when cartilage is maintained at acid pH

Bovine aggrecan was digested with bovine cathepsin D at pH 5.2 under conditions of partial digestion and the resulting aggrecan core protein fragments were separated by electrophoresis on gradient polyacrylamide gels. The fragments were characterized by their reactivity to specific antibodies and by N-terminal amino acid sequencing. It was also demonstrated that cathepsin D cleaved bovine aggrecan at five sites within the core protein, between residues Phe(342)-Phe(343) in the interglobular domain, Leu(1462)-Val(1463) between the chondroitin sulfate attachment regions 1 and 2 and Leu(1654)-Val(1655), Phe(1754)-Val(1755) and Leu(1854)-Ile(1855) that are located within the chondroitin sulfate attachment region 2 of the core protein. The time course of digestion showed that there was a continued degradation of aggrecan and there was no preferential cleavage of the core protein at any one site. It was shown that cathepsin D digested aggrecan over the pH range 5.2-6.5 resulting in the same products. When bovine cartilage was maintained in explant culture at pH 5.2 there was a rapid loss of both radiolabeled and chemical pools of sulfated glycosaminoglycans into the culture medium and this loss was inhibited by the inclusion in the medium of the aspartic proteinase inhibitor, pepstatin A. The aggrecan core protein fragments appearing in the medium of cultures maintained at pH 5.2 were characterized and it was shown that the fragments had N-terminal sequences starting at Phe(343), Ile(1855), and Val(1755) or Val(1463). This work demonstrates that cathepsin D present within the extracellular matrix of articular cartilage has the potential to contribute to the proteolytic processing of the core protein of aggrecan in this tissue.     

C-kit-immunopositive interstitial cells of Cajal in human embryonal and fetal oesophagus

Interstitial cells of Cajal (ICC) are morphologically and functionally intercalated between the elements of the enteric nervous system and the smooth muscle cells (SMCs) in the musculature of the digestive tract. Kit immunohistochemistry reliably identifies the location of these cells and provides information on changes in ICC distribution and density. Human oesophagus specimens (7 embryos, 23 fetuses at 7-27 weeks gestational age; both sexes) were exposed to Kit antibodies to determine ICC differentiation. Enteric plexuses were examined immunohistochemically by using anti-neuron-specific enolase, whereas the differentiation of SMCs was studied with antibodies against α-smooth-muscle actin and desmin. By week 7, c-kit-immunopositive cells were present along the entire oesophagus in the form of an uninterrupted layer around the myenteric plexus (MP) elements. From the beginning of the 3rd month, the number of ICC progressively decreased around the MP ganglia but increased within the muscle layers. Concomitantly, differences in the number and distribution of ICC were established in the various portions of the oesophagus: specifically, ICC were abundant in the lower portion, less numerous in the middle region and rare in the upper part. By the 5th month of development, the relationship as found in later developmental stages had been established: C-kit IR ICC were present within the circular muscle layer, within the longitudinal layer and in the connective septa surrounding the muscle bundles but were completely missing around the MP ganglia.     

Heterologous Escherichia coli Expression, Purification and Characterization of the GrmA Aminoglycoside-Resistance Methyltransferase

The mechanism of resistance to aminoglycosides based on methylation of their target, 16S rRNA, was until recently described only in antibiotic producing microorganisms. However, equivalent methyltransferases have now also been identified among numerous clinical Gram-negative pathogenic isolates. We have cloned, expressed, and purified GrmA, the aminoglycoside-resistance methyltransferase from Micromonospora purpurea, producer of gentamicin complex. Two vectors were created that express protein with an N-terminal 6× histidine tag with and without an enterokinase recognition producing proteins His₆-EK-GrmA and His₆-GrmA, respectively. The activity of both recombinant proteins was demonstrated in vivo. After optimized expression and native purification both protein variants proved to be active in in vitro methylation assays. This work lays a foundation for future detailed biochemical, structural and pharmacological studies with this member of an important group of aminoglycoside-resistance enzymes.