Ajoy Basak

The Prosegments of Furin and PC7 as Potent Inhibitors of Proprotein Convertases IN VITRO AND EX VIVO ASSESSMENT OF THEIR EFFICACY AND SELECTIVITY

All proprotein convertases (PCs) of the subtilisin/kexin family contain an N-terminal prosegment that is presumed to act both as an intramolecular chaperone and an inhibitor of its parent enzyme. In this work, we examined inhibition by purified, recombinant bacterial prosegments of furin and PC7 on the in vitro processing of either the fluorogenic peptide pERTKR-MCA or the human immunodeficiency virus envelope glycoprotein gp160. These propeptides are potent inhibitors that display measurable selectivity toward specific proprotein convertases. Small, synthetic decapeptides derived from the C termini of the prosegments are also potent inhibitors, albeit less so than the full-length proteins, and the C-terminal P1 arginine is essential for inhibition. The bacterial, recombinant prosegments were also used to generate specific antisera, allowing us to study the intracellular metabolic fate of the prosegments of furin and PC7 expressed via vaccinia virus constructs. These vaccinia virus recombinants, along with transient transfectants of the preprosegments of furin and PC7, efficiently inhibited theex vivo processing of the neurotrophins nerve growth factor and brain-derived neurotrophic factor. Thus, we have demonstrated for the first time that PC prosegments, expressed ex vivo as independent domains, can act in trans to inhibit precursor maturation by intracellular PCs.

Biosynthesis and Enzymatic Characterization of Human SKI-1/S1P and the Processing of Its Inhibitory Prosegment*

Biochemical and enzymatic characterization of the novel human subtilase hSKI-1 was carried out in various cell lines. Within the endoplasmic reticulum of LoVo cells, proSKI-1 is converted to SKI-1 by processing of its prosegment into 26-, 24-, 14-, 10-, and 8-kDa products, some of which remain tightly associated with the enzyme. N-terminal sequencing and mass spectrometric analysis were used to map the cleavage sites of the most abundant fragments, which were confirmed by synthetic peptide processing. To characterize its in vitro enzymatic properties, we generated a secreted form of SKI-1. Our data demonstrate that SKI-1 is a Ca2+-dependent proteinase exhibiting optimal cleavage at pH 6.5. We present evidence that SKI-1 processes peptides mimicking the cleavage sites of the SKI-1 prosegment, pro-brain-derived neurotrophic factor, and the sterol regulatory element-binding protein SREBP-2. Among the candidate peptides encompassing sections of the SKI-1 prosegment, the RSLK 137- andRRLL 186-containing peptides were best cleaved by this enzyme. Mutagenesis of the latter peptide allowed us to develop an efficiently processed SKI-1 substrate and to assess the importance of several P and P′ residues. Finally, we demonstrate that, in vitro, recombinant prosegments of SKI-1 inhibit its activity with apparent inhibitor constants of 100–200 nm.

The RGD Motif and the C-terminal Segment of Proprotein Convertase 1 Are Critical for Its Cellular Trafficking but Not for Its Intracellular Binding to Integrin α5β1

Cellular trafficking of subtilisin/kexin-like precursor convertases (PCs) may be regulated by a number of motifs, some of which are present within the P-domain and in the C-terminal sequence. Six of the seven known PCs contain a conserved RGD sequence within the P domain. In order to investigate the functional importance of this motif, we generated mutants of PC1 that contain a Myc tag epitope inserted between the prosegment and the catalytic subunit. Cellular expression of vaccinia virus recombinants revealed that this tag did not seem to influence the autocatalytic conversion of proPC1 into PC1 or its bioactivity. The two PC1 variants produced possess either the wild type RGD sequence or its RGE mutant. Stable transfectants of these variants in AtT20 cells revealed that similar to the wild type enzyme, PC1-RGD-Myc is sorted to secretory granules. In contrast, PC1-RGE-Myc exits the cell via the constitutive secretory pathway. In vitro, a 14-mer peptide spanning the RGD sequence of PC1, but not its RGE mutant, binds to cell surface vitronectin-binding integrins of Chinese hamster ovary cells. However, within the endoplasmic reticulum and in an RGD-independent fashion, integrin α5β1 associates primarily with the zymogens proPC1, proPC1-ΔC (missing the C-terminal 137 residues), as well as proPC2. Thus, the observed discrimination between the secretion routes of PC1-RGD and PC1-RGE does not implicate integrins such as α5β1.

Cellular Localization of Neprilysin in Mouse Bone Tissue and Putative Role in Hydrolysis of Osteogenic Peptides

The regulation of osteoblast and osteoclast metabolism is mediated by both hormones and local bone peptide factors. Peptides and hormones are under control of membrane peptidases such as Neprilysin (NEP). NEP is a widely distributed cell‐surface zinc‐metallopeptidase that is involved in the regulation of several important physiological processes by controlling the half‐life of bioactive peptides. Although NEP is known to be present in skeletal tissues, neither its cellular localization nor its function have been established. To address this question, we examined NEP distribution in bones of postnatal mouse. In situ hybridization (ISH) and immunohistochemistry showed that NEP messenger RNA (mRNA) and protein are associated with bone‐forming cells including presumptive osteoblast precursors, preosteoblasts, osteoblasts, and osteocytes. NEP levels in newborn and adult mice bones also were compared by immunoblotting. Higher amounts of NEP immunoreactivity were observed in newborn as compared with adult bones, suggesting a relationship between NEP expression and bone growth. To further explore this hypothesis, we monitored in vitro NEP proteolytic activity using a series of synthetic osteogenic peptides such as parathyroid hormone‐related peptide 1–43 (PTHrP1–34), osteostatin (PTHrP107–139), osteogenic growth peptide (OGP), calcitonin, α‐calcitonin gene‐related peptide (α‐CGRP), and PTH1–34. Except for PTH1–34, all peptides were found to be NEP substrates.

Detection of endopeptidase activity and analysis of cleavage specificity using a radiometric solid-phase enzymatic assay

A radiometric procedure to detect the presence of proteolytic enzymes and analyze their substrate specificity is described. The enzymatic activity is first measured by the release into solution of a radiolabeled reporter group from an immobilized peptidyl substrate. Two peptidyl substrates encompassing multiple cleavage sites, a derivative of Leu-enkephalin and a peptide related to the bait region of human alpha 2-macroglobulin, are prepared and linked via a spacer molecule to an insoluble support. The labeled peptides released are then separated by high-performance liquid chromatography. The position of the released peptides upon chromatography allows direct identification of the sites of cleavage. The assay, using a radioactive iodinated tyrosine residue as reporter group, is extremely sensitive (less than 0.02 pg/ml of trypsin), reproducible, and easy to perform while yielding unambiguous identification of the sites of cleavage. This assay can be used to detect the presence of enzymatic activities and/or of enzyme inhibitors. Furthermore, it can be easily adapted to detect from a variety of sources all four classes of enzymes known by using appropriate peptidyl substrate sequences, buffer, pH, and incubation conditions.

Affinity purification of proteinases by a combination of immobilized peptidyl aldehyde and semicarbazone

D-Phe-Phe-argininal semicarbazone and Tyr-Gly-Gly-Phe-Leu-Arg-argininal semicarbazone were prepared using the solution phase synthesis method and characterized by mass spectrometry and nuclear magnetic resonance spectroscopy. The tripeptide and heptapeptide semicarbazones were individually immobilized on affi-Gel 15 resulting in two affinity columns called S3 and S7, respectively. A third affinity column was obtained by hydrolysing the semicarbazone moiety in column S3 to aldehyde (column A3). Serine proteinases such as trypsin or rat plasma kallikrein almost quantitatively bind to either S3 or A3 affinity columns. Under optimized conditions, more than 97% of trypsin bound to both columns S3 and A3. At a lower ionic strength and higher pH, 80-85% of rat plasma kallikrein bound to the same columns. Elution of both enzymes was achieved using mild conditions at near neutral pH and in the presence of a small amount of denaturant. Both proteinases were identified and characterized by high-performance liquid chromatography, sodium dodecylsulphate polyacrylamide gel electrophoresis and by their substrate specificity and inhibition profiles. A single purification (six-to seven-fold) step using either column S3 or A3 allowed the preparation of pure trypsin from commercial sources. Starting from rat plasma partially purified by a phenyl boronate column, fractionation on the S3 column allowed approximately an 87-fold purification of rat plasma kallikrein. However, serial purification of rat plasma kallikrein on column S7 followed by column A3 resulted in a purification factor of about 455.

Synthesis of the segment (11-23) located in the first tandem repeat of plasma kallikrein: comparative binding studies of this and another segment (328-343) to high-molecular-mass kininogen.

The synthesis of porcine plasma kallikrein (pPK) segment (11-23), of sequence Phe-Phe-Arg-Gly-Gly-Asp-Val-Ser-Ala-Met-Tyr-Thr-Pro, present in the first tandem repeat sequence of the regulatory chain of PK, has been accomplished following the peptide fragments (5 + 4 + 4) condensation strategy in solution, as well as by fluorenylmethoxycarbonyl solid-phase chemistry. This and another synthetic PK segment of residues (328-343) present in the fourth tandem repeat sequence [Cys(ACM)-Ser-Leu-Arg-Leu-Ser-Thr-Asp-Gly-Ser-Pro-Thr-Arg-Ile-Thr-Tyr] and synthesized by a solid-phase method, were fully characterized by 1H nuclear magnetic resonance, fast atom bombardment mass spectrometry, amino acid composition and reversed-phase high-performance liquid chromatography. Proteolysis of these peptides by either rat PK (rPK) or trypsin resulted in cleavages between Arg decreases Gly for pPK (11-23) and between Arg decreases Leu and Arg decreases Ile for rPK (328-343). Kinetic studies revealed that for peptide pPK (11-23), the catalytic efficiency (kcat/Km) of rPK is congruent to 9-fold higher than that of trypsin, but for the other peptide, rPK (328-343), kcat/Km of trypsin is congruent to 49-fold higher than that of rPK. The facile cleavage of pPK (11-23) by rPK confirms the Arg13 decreases Gly14 position as the site of autolytic degradation of PK and also explains its special preference for Phe-Phe-Arg sequence.