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. 2016 Aug 19;291(34):17706-16.
doi: 10.1074/jbc.M116.743237. Epub 2016 Jun 29.

Complex Formation of Human Proelastases With Procarboxypeptidases A1 and A2

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Free PMC article

Complex Formation of Human Proelastases With Procarboxypeptidases A1 and A2

András Szabó et al. J Biol Chem. .
Free PMC article

Abstract

The pancreas secretes digestive proenzymes typically in their monomeric form. A notable exception is the ternary complex formed by proproteinase E, chymotrypsinogen C, and procarboxypeptidase A (proCPA) in cattle and other ruminants. In the human and pig pancreas binary complexes of proCPA with proelastases were found. To characterize complex formation among human pancreatic protease zymogens in a systematic manner, we performed binding experiments using recombinant proelastases CELA2A, CELA3A, and CELA3B; chymotrypsinogens CTRB1, CTRB2, CTRC, and CTRL1; and procarboxypeptidases CPA1, CPA2, and CPB1. We found that proCELA3B bound not only to proCPA1 (KD 43 nm) but even more tightly to proCPA2 (KD 18 nm), whereas proCELA2A bound weakly to proCPA1 only (KD 152 nm). Surprisingly, proCELA3A, which shares 92% identity with proCELA3B, did not form stable complexes due to the evolutionary replacement of Ala(241) with Gly. The polymorphic nature of position 241 in both CELA3A (∼4% Ala(241) alleles) and CELA3B (∼2% Gly(241) alleles) points to individual variations in complex formation. The functional effect of complex formation was delayed procarboxypeptidase activation due to increased affinity of the inhibitory activation peptide, whereas proelastase activation was unchanged. We conclude that complex formation among human pancreatic protease zymogens is limited to a subset of proelastases and procarboxypeptidases. Complex formation stabilizes the inhibitory activation peptide of procarboxypeptidases and thereby increases zymogen stability and controls activation.

Keywords: complex; elastase; metalloprotease; pancreas; pancreatitis; proteolytic enzyme; serine protease; zymogen activation.

Figures

FIGURE 1.
FIGURE 1.
Autolytic cleavage sites in the activation peptide and N-terminal region of human proCELA3A and proCELA3B. The activation peptides (Tyr18-Arg28) are highlighted in gray. The activation site Arg28 is emboldened. See text for details.
FIGURE 2.
FIGURE 2.
Binary complex formation of human pancreatic protease zymogens. Qualitative binding assays using His-tagged proelastases (proCELA, including proCELA2A, proCELA3A, and proCELA3B), His-tagged chymotrypsinogens (CTRB1, CTRB2, CTRC, and CTRL1), and non-tagged procarboxypeptidases (proCP, including proCPA1, proCPA2, and proCPB1) were performed as described under ”Experimental Procedures.“ The numbers above the lanes indicate the fractions collected after elution from the nickel column. None of the chymotrypsinogens formed complexes; representative negative results are shown for the CTRC zymogen only. Representative gel pictures of two binding experiments are shown.
FIGURE 3.
FIGURE 3.
Binding of proCELA3B to proCPA1 (A) and proCPA2 (B). Equilibrium binding assays using purified proenzymes were carried out as described under ”Experimental Procedures.“ The free (unbound) proCPA concentrations were plotted as a function of the total proCELA3B concentration. Data points represent the average of three replicates ± S.E. Error bars may be smaller than symbol sizes. Data were fitted globally, the KD values and the error of the fits are indicated. The catalytically inactive S217A mutant of proCELA3B was used.
FIGURE 4.
FIGURE 4.
Binding of proCELA2A and proCELA3A to proCPA1 (A) and proCPA2 (B). Equilibrium binding assays using purified proenzymes were carried out as described in the legend to Fig. 3 and under ”Experimental Procedures.“ See text for the error of the fits. For comparison, binding data for wild-type proCELA3B from Fig. 3 are also indicated. Note that KD values in the micromolar range should be considered estimates. The catalytically inactive S217A mutant of proCELA3A was used.
FIGURE 5.
FIGURE 5.
Structural determinants of defective proCELA3A binding to proCPA1 and proCPA2. A, the positions of amino acid differences between proCELA3A and proCELA3B were mapped to the binding interface between bovine proCPA and proproteinase E. For clarity, chymotrypsinogen C was omitted from the ternary complex (Protein Data Bank file 1PYT). Amino acid residues at or near the interface are indicated. B, binding of proCELA3B triple mutant S77R,S78D,W79L and single mutants S197T and A241G to proCPA1 and proCPA2. C, binding of proCELA3A mutant G241A to proCPA1 and proCPA2. Qualitative binding experiments using conditioned media with His-tagged proelastases and non-tagged procarboxypeptidases were carried out as described under ”Experimental Procedures.“ The numbers above the lanes indicate the fractions eluted from the nickel column. Representative gel pictures of two experiments are shown.
FIGURE 6.
FIGURE 6.
Binding of proCELA3A variant G241A and proCELA3B variants W79R and A241G to proCPA1 (A) and proCPA2 (B). Equilibrium binding assays using purified proenzymes were carried out as described in the legend to Fig. 3 and under ”Experimental Procedures.“ For comparison, binding data for wild-type proCELA3A and proCELA3B from Figs. 3 and 4 are also indicated. Data points represent the average of three replicates ± S.E. Error bars may be smaller than symbol sizes. See text for the error of the fits. Note that KD values in the micromolar range should be considered estimates. The proCELA3A and proCELA3B constructs used contained the S217A catalytic mutation.
FIGURE 7.
FIGURE 7.
Effect of wild-type proCELA3B and mutant A241G on the activation of proCPA1 (A) and proCPA2 (B) by trypsin and CTRC. Procarboxypeptidases at 200 nm concentration were incubated at 37 °C in the absence (control) or presence of 2 μm wild-type proCELA3B or A241G mutant in 0.1 m Tris-HCl (pH 8.0), 1 mm CaCl2, and 0.05% Tween 20 (final concentrations) in 200 μl final volume. Where indicated by arrows, 5 nm human cationic trypsin and 5 nm human CTRC were added. At given times, 8- (CPA1) or 15-μl (CPA2) aliquots were withdrawn and carboxypeptidase activity was measured as described under ”Experimental Procedures.“ Data points represent the average of three replicates ± S.E. Error bars may be smaller than symbol sizes. The catalytically inactive S217A mutants of proCELA3B and proCELA3B A241G were used.
FIGURE 8.
FIGURE 8.
Effect of wild-type proCELA3B and mutant A241G on the binding of the inhibitory activation peptides of proCPA1 (A) and proCPA2 (B). Procarboxypeptidases were incubated at 2 μm concentration with 50 nm human cationic trypsin at 37 °C in 0.1 m Tris-HCl (pH 8.0), 15 mm NaCl, 1 mm CaCl2, and 0.05% Tween 20 (final concentrations) in 100 μl final volume. After 30 min incubation, activated proenzymes were diluted to the indicated concentrations in 100 μl final volume with assay buffer (0.1 m Tris-HCl (pH 8.0), 150 mm NaCl, 1 mm CaCl2, and 0.05% Tween 20) and incubated for 10 min at 22 °C in the absence (control) or presence of 500 nm wild-type proCELA3B or A241G mutant. Carboxypeptidase activity was then measured by adding 10 μl of N-[4-methoxyphenylazoformyl]-l-phenylalanine substrate to 60 μm final concentration, as described under ”Experimental Procedures.“ Carboxypeptidase activities were converted to free CPA1/CPA2 concentrations by dividing the activity values with the slope of the linear concentration-activity plots of CPA1/CPA2 fully activated with trypsin plus CTRC (see supplemental Fig. S1 in Ref. 2). Data points represent the average of three replicates ± S.E. Error bars may be smaller than symbol sizes. Data points were fitted to the equation: y = (−K + sqrt(K∧2 + 4Kx))/2, where K is the equilibrium dissociation constant. The variable x is the total concentration of trypsin-activated proCPA1/proCPA2 present in the reaction and y is the concentration of free CPA1/CPA2 in equilibrium. The catalytically inactive S217A mutants of proCELA3B and proCELA3B A241G were used.

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