Structure and Function Studies of Hsp47: A Collagen-Specific Molecular Chaperone

Abstract

<p>Heat shock protein 47 (Hsp47) is an ER resident protein and is considered to be a molecular chaperone specific for collagen. Although Hsp47 has been shown to be necessary for collagen biosynthesis, its exact roles(s) as a chaperone for collagen is/are unknown. Available evidence on type I collagen shows that Hsp47 binds to the nascent proα and proα2 chains and remains bound during triple-helix formation and subsequent export from the ER. Upon entering the cis-Golgi network, Hsp47 is thought to release procollagen and is subsequently returned to the ER. With a view to understanding the exact role of Hsp47 in collagen biosynhesis, we undertook biophysical and biochemical characterization of Hsp47. This involved purification of recombinant Hsp47 using the IMPACT™ T7 system. Purified Hsp47 existed as both a monomer and a trimer, and underwent pH-induced conformational changes that correlated to its collagen-binding activity. Hsp47 was shown to effectively inhibit type I collagen fibrillogenesis in vitro. This inhibition of fibre formation provided a novel assay for Hsp47 activity, as well as support for an in vivo role for Hsp47 in preventing collagen aggregation in the ER. Finally, in an attempt to map the binding site(s) on the collagen molecule, assays were designed to analyze the interaction of radio-labelled Hsp47 with CNBr fragments derived from type I and type II collagen. The results showed that Hsp47 preferentially binds to peptides from the N-terminal region of collagen in their triple-helical conformation</p>