Molecular characterization and structure determination of human ADAMTSL4

Abstract

The thrombospondin type 1 repeat (TSR) is an ancient extracellular protein domain that is commonly found in invertebrate and vertebrate proteins. The ADAMTSL4 protein, also known as TSRC1, belongs to the TSR superfamily and has multiple thrombospondin repeats, most of which are clustered at the C-terminus. It has been reported that some TSP1 domain-containing proteins, e.g. thrombospondin 1 and thrombospondin 2, could induce apoptosis of endothelial cells but it is not clear how these proteins operate in death pathways. Our recent work shows that mutations in ADAMTSL4 are responsible for autosomal-recessive isolated ectopia lentis, i.e. abnormal positioning of the lens of the eye and affect the development of the zonular fiber. However, little is known about the function of ADAMTSL4. To shed more light on the function of the ADMTSL4 and its roles in different tissues we extended our work to carry out molecular characterization of this gene and its variants. We have obtained cDNA clones encoding the full ADAMTSL4 protein and its truncated isoform. Both are subcloned into the pET28a vector for expression in E. coli. In anticipation of possible expression challenges in this host, e.g. formation of inclusion bodies or lack of expression, we subcloned each fragment into the yeast vector, pYES2. The gene in both cases has been fused in frame with a region encoding an N-terminal His-Tag to facilitate the purification of the recombinant protein. DNA analysis indicates that each fragment has been correctly cloned into the pYES2 vector. Each construct was transformed into Saccharomyces cerevisiae for protein expression. Our preliminary analysis indicates that one of the genes is expressed in S. cerevisiae but at a very low level. Work to optimize the expression of ADAMTSL4 in yeast as well as in E. coli is in progress. We also extracted the seven domains of the ADAMTSL4 using PCR. Each domain was subcloned into the E. coli overexpression vector, pET28a. Expression studies of all the constructs have shown that the seven domains have been successfully overexpressed in E. coli. The overexpression was confirmed using Western blot techniques. The recombinant protein of each domain is purified for NMR and x-ray studies.