Molecular Characterization, Enzymatic Analysis, and Purification of Murine Proprotein Convertase-1/3 (PC1/PC3) Secreted from Recombinant Baculovirus-Infected Insect Cells

The cDNA of a tomato subtilase designated LeSBT1 was cloned from a tomato flower cDNA library. The deduced amino acid sequence indicated for LeSBT1 the structure of a prepro-protein targeted to the secretory pathway by virtue of an amino-terminal signal peptide. LeSBT1 was expressed in the baculovirus/insect cell system and a processed 73-kDa form of LeSBT1, lacking both signal peptide and prodomain, was purified to homogeneity from culture supernatants. This 73-kDa LeSBT1, however, lacked proteolytic activity. Zymogen activation to yield 68-kDa LeSBT1 required the additional processing of an amino-terminal autoinhibitory peptide in a strictly pH-dependent manner. Mature 68-kDa LeSBT1 showed highest activity at acidic pH consistent with its presumed localization in the apoplast of the plant cell. In comparison to other plant subtilases, LeSBT1 exhibited a narrower substrate specificity in that it cleaves only polypeptide substrates preferentially but not exclusively carboxyl-terminal of glutamine residues. The possible involvement of LeSBT1 in selective proprotein processing is discussed with reference to the related mammalian proprotein convertases.

Section: Discussionmentioning

confidence: 79%

Section: Discussionmentioning

confidence: 92%

LeSBT1, a Subtilase from Tomato Plants

Janzik

Macheroux

Amrhein

et al. 2000

“…Expression and Purification of proPC1/3-To obtain large amounts of recombinant enzymatically active PC1/3, we have used the baculovirus/insect cell expression system and found that enzymatically active PC1/3 was secreted into the medium from which it can be purified, yielding 1-2 mg/liter of medium of highly pure 85-or 71-kDa enzyme (23). Under these expression conditions, enzymatically inactive PC1/3-related proteins also accumulate intracellularly in inclusion bodies as disulfidelinked aggregates at a level of about 10 g/1 ϫ 10 6 cells or approximately 50 mg/liter of cell culture (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…The pelleted cells were frozen at Ϫ20°C until necessary, whereas the resulting supernatant was decanted and centrifuged again. The soluble and secreted PC1/3 was purified to homogeneity from the infected insect cell supernatant by polyethylene glycol precipitation followed by immobilized lectin, hydroxylapatite, and gel permeation chromatography as described previously (23). The resulting purified 71-kDa enzyme is homogeneous and is more than 95% pure as judged by SDS-PAGE analysis and exhibits a K m of 8.1 Ϯ 0.6 M for the pGlu-Arg-Thr-Lys-Arg-MCA substrate and a specific activity of 2.95 M/h/mg of amino-4-methylcoumarin-released (23).…”

Section: Methodsmentioning

confidence: 99%

Proprotein Convertase PC1/3-related Peptides Are Potent Slow Tight-binding Inhibitors of Murine PC1/3 and Hfurin

Boudreault

Gauthier

Lazure

1998

Self Cite

The proprotein convertase PC1/3 belongs to the subtilisin/kexin-like endoprotease family and is synthesized as a preproenzyme. To investigate the function of its propeptide, murine proPC1/3 and preproPC1/3 were isolated from the inclusion bodies of recombinant preproPC1/3 baculovirus-infected insect cells, rendered soluble with 6 M guanidine HCl and 20 mM dithiothreitol, and purified by gel filtration and metal-binding affinity chromatography. Two NH 2 -terminal fragments containing the complete propeptide 1-84 region were obtained after CNBr cleavage, purified, and chemically characterized. Progress curve kinetic analysis with enzymatically active murine 71-kDa PC1/3 or 50-kDa human furin demonstrated that both fragments were potent slow tight-binding inhibitors of either enzyme with K i in the low nanomolar range. Additional cleavages at Trp residues yielded fragment 9 -71 , which no longer represents a potent inhibitor. Upon incubation at pH 5.5 in the presence of excess 71-kDa murine PC1/3, NH 2 -terminal fragment 1-98 is cleaved at two sites, as revealed through Western blotting using NH 2 -terminal-directed PC1/3 antibodies. Finally, murine PC2 is inhibited by the proPC1/ 3 1-98 peptide, albeit at a much lesser extent with a micromolar K i and in a strictly competitive manner. These results suggest that the proregion of PC1/3 is an important feature in regulating its activity.

“…Modifications to the original recombinant virus described in Boudreault et al (21) include substitution of the signal peptide of mPC1/3 by the one of the viral glycoprotein 67 (gp67) to enhance the secretion of the recombinant enzyme into the medium of Spodoptera frugiperda cells. Once expressed, the enzyme is recovered and purified as previously described (20,21). The recombinant soluble (COOH terminus truncated) human furin is obtained from the medium of Sf9 insect cells (13).…”

Section: Expression and Purification Of Recombinant Mpc1/3 And Humanmentioning

confidence: 99%

Single Amino Acid Substitution in the PC1/3 Propeptide Can Induce Significant Modifications of Its Inhibitory Profile toward Its Cognate Enzyme

Rabah

Gauthier

Wilkes

et al. 2006

Self Cite

The proprotein convertase PC1/3 is synthesized as a large precursor that undergoes proteolytic processing of the signal peptide, the propeptide and ultimately the COOH-terminal tail, to generate the mature form. The propeptide is essential for protease folding, and, although cleaved by an autocatalytic process, it remains associated with the mature form acting as an auto-inhibitor of PC1/3. To further assess the role of certain residues in its interaction with its cognate enzyme, we performed an alanine scan on two PC1/3 propeptide potential cleavable sites ( 50 RRSRR 54 and 61 KR 62 ) and an acidic region 65 DDD 67 conserved among species. Upon incubation with PC1/3, the ensuing peptides exhibit equal inhibitory potency, lower potency, or higher potency than the wild-type propeptide. The K i values calculated varied between 0.15 and 16.5 nM. All but one mutant exhibited a tight binding behavior. To examine the specificity of mutants, we studied their reactivity toward furin, a closely related convertase. The mutation of certain residues also affects the inhibition behavior toward furin yielding propeptides exhibiting K i ranging from 0.2 to 24 nM. Mutant propeptides exhibited against each enzyme either different mode of inhibition, enhanced selectivity in the order of 40-fold for one enzyme, or high potency with no discrimination. Hence, we demonstrate through single amino acid substitution that it is feasible to modify the inhibitory behavior of propeptides toward convertases in such a way as to increase or decrease their potency, modify their inhibitory mechanisms, as well as increase their selectivity.One of the most common methods used by cells to diversify the pool of their biologically active molecules is protein processing. Indeed, numerous secreted proteins are synthesized first as an inactive precursor, which is rendered biologically active upon cleavage at clusters of basic residues. Members of a family of proteins named proprotein convertases (PCs) 3 primarily perform this cleavage. To date, seven members were described: furin, PC1/3, PC2, PACE4, PC4, PC5/6, and PC7/ PC8/lymphoma proprotein convertase. Some of them are ubiquitously expressed such as furin, PACE4, and PC7, whereas others exhibit a more restricted expression pattern such as PC1/3 and PC2, which are solely present in endocrine and neuroendocrine tissues, and PC4, which is expressed only in germ cells. However, all of them belong to the larger family of serine proteases and are structurally related to bacterial subtilisin and yeast kexin (reviewed in Ref. 1). In terms of biological activities, numerous transfection experiments using recombinant enzymes and substrates, generation of knock-out animals as well as human cases of convertase deficiency pointed out the importance of convertases in crucial biological processes such as patterning during embryogenesis, angiogenesis, prohormone processing, tissue remodeling, and complement activation (2). Furthermore, some members are also implicated in many disease states, because they are able to...