Characterization of Yeast Aspartic Protease 3 (A novel basic-residue specific prohormone processing enzyme)
Abstract
Yeast aspartic protease 3 (YAP3p). was first cloned. based on its ability to correctly process pro-a-mating factor at specific paired-basic residue cleavage sites. Thespecificity of this aspartic protease for basic residue cleavage sites of prohormones renders it a member of a novel sub-class of aspartic proteases that may be involved in prohormone and proneuropeptide processing in general. Y AP3p was first overexpressed, purified and characterized from the cellular extract of induced yeast. The cellular form of YAP3p was characterized as a -70kDa glycoprotein and was shown to cleave pro-opiomelanocortin and its fragments at specific paired-basic residue cleavage sites in vitro. Deletion of 0.2kb fragment from the 3' end of the YAP3 cDNA resulted in the expression of a carboxy-terminally truncated protein which had a hydrophobic domain and a putative glycophosphatidylinositol membrane anchor removed. This truncated YAP3p was secreted into the growth media, purified and characterized as ~150-180kDa and ~90kDa hyperglycosylated glycoproteins with optimum enzymatic activity between pH 4.0-4.5.Removal of the N-linked sugars from the hyperglycosylated y AP3p by endoglycosidase H resulted in the reduction in size of both forms to a single ~65kDa protein. In comparison to the calculated isoelectric point (pI) of the truncated y AP3p of 4.0, a pI value of ~4.5 was determined experimentally by isoelectric focussing gel electrophoresis. A Q(sub 10) of 1.95 for YAP3p was calculated when the dependence of YAP3p activity on temperature was determined. Secreted YAP3p was shown to cleave at mono-, paired- and tetra-basic residue cleavage sites of many prohormone substrates. Analysis of the kinetic parameters for the cleavage of these substrates demonstrated that Y AP3p preferred paired- versus mono-basic sites, with the most efficient cleavage being at the tetra-basic residue site ofACTH.
Document Details
- Document Type
- Technical Report
- Publication Date
- May 16, 1995
- Accession Number
- AD1011442
Entities
People
- Niamh X. Cawley
Organizations
- Uniformed Services University of the Health Sciences