Characterisation of glycoprotein III from chromaffin granules: association with lipid and interaction with components involved in the processing of prohormones.

Abstract

Chromaffin granules are vesicles from the adrenal medulla that have a specialised role in the regulated secretion of catecholamines and peptide hormones. These subcellular organelles are involved in hormone biosynthesis, storage and secretion. One glycoprotein component of bovine-derived chromaffin granules is GpIII, a homolog of the widely distributed protein clusterin. This glycoprotein has been assigned a number of interesting roles, all related to its protein and lipid-binding properties. In chromaffin granules, GpIII can be found in both the membrane and soluble fractions of chromaffin granule lysate. In the soluble fraction this protein is present as part of high molecular weight aggregates along with glycoproteins H (the endoproteases prohormone convertase 3/1 and prohormone convertase 2) and J/K (carboxypeptidase H). Preliminary studies indicated that lipid was present in these complexes. The aim of this thesis is to further characterise the GpIII-containing aggregates of chromaffin granules. The lipid component of these aggregates was investigated, and found to contribute a significant proportion of the mass. These aggregates were shown to resemble high density lipoprotein particles of ~30nm diameter and to have a protein/phospholipid/cholesterol ratio of 1:0.93:0.08. GpIII was found to bind specifically to cholesterol/cholesterol esters, presumably through interactions of one or more amphipathic α-helical regions within the protein. This association of GpIII with lipids offers an explanation as to why no differences in the protein purified from membranes and the soluble lysate could be determined. The glycoprotein components GpH, J and K are also present in chromaffin granules in membrane-associated and a soluble forms. The mechanism of association of GpJ/K with the GpIII-containing aggregates was investigated to determine the role of a carboxyl terminal amphipathic α-helix in binding. This region is preceded by a pair of basic residues that may represent a putative site for endoprotease cleavage. The presence or absence of this region in CpH from various chromaffin granule fractions, including GpIII-associated GpJ/K was determined. The interactions of the processing enzymes, in particular CpH, with the GpIII aggregate have been investigated. It was shown that while a sequence at the extreme carboxy-terminus could be found in both membrane and soluble pools, this region of CpH was required for its association with GpIII. A model was developed whereby the GpIII aggregate mimics the close association of GpIII with the prohormone processing enzymes present in the membrane of chromaffin granules. Addition of purified recombinant PC3/1 (kex 2-like endoproteases) resulted in increased activity, providing indirect evidence that proteolytic cleavage of the carboxy-terminus of GpJ/K may stimulate activity and release the enzyme from the GpIII aggregate. It is proposed that GpIII-containing complexes mimic interactions occurring between these glycoproteins in the membrane fraction and may therefore have a role in sorting of prohormone processing enzymes. The controlled release of processing enzymes from these soluble aggregates may be important for the regulation of activity.