Oral Presentation Matrix Biology Society of Australia and New Zealand Annual Meeting 2012

Elucidation of alternatively spliced forms of perlecan (#27)

Bonny M Tsoi 1 , Megan S Lord 1 , MoonSun Jung 1 , Bill Cheng 1 , J Guy Lyons 2 , John M Whitelock 1
  1. UNSW, Kensington, NSW, Australia
  2. Discipline of Dermatology, Bosch Insititute, Sydney Cancer Centre, University of Sydney, Sydney, NSW, Australia

Perlecan, a ubiquitous and modular heparan sulfate (HS) proteoglycan, has been hypothesised to have roles in a wide variety of biological and pathological processes. The perlecan gene, HSPG2, is under the control of a single promoter, which is regulated by transcription factors downstream of TGFb signalling.  Shorter forms of the HSPG2 gene have been reported in the NCBI database, but have not been followed up with respect to molecular structure and potential biological functions.  We have shown previously that human mast cells produce perlecan with a Mr of 640 kDa as well as smaller fragments with 300 and 130 kDa. N-terminal sequencing of the 130 kDa band showed that it was a mix of peptides corresponding to sequences in domains II, III, IV and V supporting the idea of proteolytic processing of the perlecan protein core.  These fragments were reactive against domain V antibodies in ELISAs, using a range of domain-specific antibodies, indicated that this domain was produced at relatively higher amounts compared to other domains.  In addition, a set of domain specific quantitative PCR experiments were performed to compare the amplification of the various domains using the same primer sets between the mast cells and endothelial cells.  Results from these experiments demonstrated endothellial cells expressed relatively more transcripts for all domains, whereas mast cells produced more transcripts for domain V than either domains I or III.   This data supports the idea of splicing events favouring the appearance of mRNA containing domain V but further experiments are required to identify the potential splice sites that are hypothesised to be in domains I and IV.  Our hypothesis is mast cells produce shorter forms of perlecan that contain domain V via mRNA alternative splicing, which has downstream biological effects on cell adhesion,  migration and proliferation that are all fundamental events involved in processes where mast cells have major roles, such as angiogenesis and matrix turnover.