In Vitro Characterization of Proglucagon Cleavage by PCI

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Bonic, Anela
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The activation of many biologically active peptides occurs through the sequential action of propeptide converting enzymes on less active precursors called propeptides. Propeptide converting enzymes recognize and cleave their respective propeptide substrates on the carboxy-terminal side of specific cleavage sites identified most commonly by dibasic residues and less often by tetrabasic and monobasic residues. Identification of a furin/propeptide convertase (furin/PC) family of endoproteases has led to in vivo and in vitro studies aimed at understanding the mechanism of selective precursor processing at basic residues.|To understand how enzymes function, their kinetic properties have to be analyzed under controlled conditions. It is thought that structural information, including the basic amino acids at these cleavage sites, is involved in determining the specificity of the furin/PCs. I hypothesize that cleavage specificity of a member of the furin/PC family can be quantitatively characterized in vitro with a full-length propeptide substrate.|Milligram quantities of methionylated proglucagon (Met-PG), its N-terminal derivative glicentin (Met-Gli) and the C-terminal derivative major pro glucagon factor (Met-MPGF) were successfully expressed and purified for use as model substrates to characterize the cleavage specificity of PCI, an endoprotease of the furin/PC family of enzymes. Under conditions of the in vitro assay, PCI cleaves Met-PG at two sites to produce glicentin, glucagon-like peptide-1 (GLP-1) and an extended form of GLP-2. PCI does not cleave Met-Gli, but does cleave Met-MPGF at a single site to produce Met- GLP-1 and an N-terminally extended version of GLP-2.|To simplify kinetic analysis of Met-PG processing by PCI, Met-MPGF cleavage at the C-terminal end of the GLP-1 sequence was examined. The ability of PCI to cleave this site was compared to its ability to cleave the fluorogenic pentapeptide substrate Pyr- RTKR-MCA. By incubating PCI with Pyr-RTKR-MCA the following values were obtained: kcat = 0.03 sec*', KM =110 pM and kca/KM = 300 sec*'M*'. Upon examination of PCI-mediated cleavage of Met-MPGF to produce GLP-1, the following values were obtained: kcat = 0.1 sec*', KM = 55 pM and kca/KM = 2,400 sec 'M*'. These data suggest that Met-MPGF is a better PCI substrate than the pentapeptide, and are consistent with the hypothesis that signals such as secondary or tertiary structural elements, in addition to dibasic cleavage sites, could be involved in facilitating recognition and cleavage of selected sites by members of the furin/PC family of enzymes.
Creighton University
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