Cytosolic carboxypeptidase 5 removes α- and γ-linked glutamates from tubulin

Abstract

Cytosolic carboxypeptidase 5 (CCP5) is a member of a subfamily of enzymes that cleave C-terminal and/or side chain amino acids from tubulin. CCP5 was proposed to selectively cleave the branch point of glutamylated tubulin, based on studies involving overexpression of CCP5 in cell lines and detection of tubulin forms with antisera. In the present study, we examined the activity of purified CCP5 toward synthetic peptides as well as soluble α- and β-tubulin and paclitaxel-stabilized microtubules using a combination of antisera and mass spectrometry to detect the products. Mouse CCP5 removes multiple glutamate residues and the branch point glutamate from the side chains of porcine brain α- and β-tubulin. In addition, CCP5 excised C-terminal glutamates from detyrosinated α-tubulin. The enzyme also removed multiple glutamate residues from side chains and C termini of paclitaxel-stabilized microtubules. CCP5 both shortens and removes side chain glutamates from synthetic peptides corresponding to the C-terminal region of β3-tubulin, whereas cytosolic carboxypeptidase 1 shortens the side chain without cleaving the peptides' γ-linked residues. The rate of cleavage of α linkages by CCP5 is considerably slower than that of removal of a single γ-linked glutamate residue. Collectively, our data show that CCP5 functions as a dual-functional deglutamylase cleaving both α- and γ-linked glutamate from tubulin.

Keywords: Carboxypeptidase; Cytosolic Carboxypeptidase; Enzymes; Metalloenzymes; Peptidases; Tubulin.

FIGURE 1.

Purification of recombinant CCP1 and CCP5. A, immunoblot analysis of metal affinity column fractions obtained following purification. The Sf9 insect cells were infected with WT baculovirus or with baculovirus expressing His-tagged CCP1 or His-tagged CCP5. Aliquots of cell extracts (input), non-bound fraction (flow-through; FT), and elute with 80 mm imidazole were separated by SDS-PAGE and probed with tetra-His antibody. B, protein staining of the samples used for the immunoblot. The expected molecular masses of CCP1 and CCP5 are 130 and 90 kDa, respectively. M, marker.

FIGURE 2.

Characterization of CCP5 enzymatic activity toward purified porcine brain tubulin. Purified brain tubulin was incubated with purified CCP5 or control eluates (Ctl) at 37 °C for 1 h at different conditions, slot-blotted into the nitrocellulose membrane, and then probed with GT335 and α-tubulin antibodies. A, C, and E, representative slot blots for GT335 and α-tubulin. B, D, and F, quantification of replicate slot blots. The GT335 band densities were normalized with the corresponding α-tubulin bands. CCP5 activity was measured as loss of GT335 signal. Error bars, S.E. (n = 3). A and B, effect of pH on CCP5 activity toward brain tubulin. C and D, effect of NaCl on CCP5 activity toward brain tubulin. E and F, reaction curve for CCP5 activity toward brain tubulin.

FIGURE 3.

Effect of paclitaxel on CCP1 and CCP5 enzymatic activity toward purified porcine brain tubulin. Purified brain tubulin (50 μg/ml final concentration) was preincubated in the presence or absence of 5 μm paclitaxel (Taxol) for 15 min at 37 °C, and then CCP1, CCP5, or control eluate (Ctl) was added and incubated for a further 1 h at 37 °C. Samples were slot-blotted into nitrocellulose membrane and probed with GT335 antibody. A, representative slot blots. B, densitometric analysis of GT335. The GT335 band densities were normalized with the corresponding α-tubulin bands. Error bars, S.E. (n = 3). *, p ≤ 0.05; **, p ≤ 0.01 using Student's t test. NS, not significant.

FIGURE 4.

CCP5 enzymatic activity toward polymerized tubulin. Polymerized microtubules from MDA-MB-231 cells were prepared on plates using microtubule-stabilizing buffer containing 5 μm paclitaxel in the absence of GTP. For immunostaining with tyrosinated tubulin and Δ2-tubulin antibodies, microtubules were treated with carboxypeptidase A1 (40 ng/ml) to convert tyrosinated into detyrosinated tubulin. After washes, microtubules were incubated with CCP5 or control eluate for 2 h and then processed for immunocytochemistry.

FIGURE 5.

Brain tubulin processing by purified CCP5. Purified porcine brain tubulin was incubated at 37 °C for 1 h with purified CCP5 or control eluates. After incubation, tubulin was separated by SDS-PAGE, dissected out from the gel, digested with CNBr, and analyzed by mass spectrometry. A, representative spectra of the C-terminal region of the α1a/α1b/α3 form of porcine α-tubulin after incubation with control eluate (top) or after incubation with CCP5 (bottom). The theoretical monoisotopic mass of the C-terminal fragment with no Glu side chain residues is 2859.19 (sequence AALEKDYEEVGVDSVEGEGEEEGEEY). The molecular mass of a Glu residue is 129 Da. B, representative spectra of the C-terminal region of β2b form of porcine β-tubulin after incubation with control eluate (top) or with CCP5 (bottom). The theoretical monoisotopic mass of the C-terminal fragment with no Glu side chain residues is 3465.36 (sequence NDLVSEYQQYQDATADEQGEFEEEEGEDEA). C, representative spectrum of the C-terminal region of α4a-tubulin after incubation with control eluate (top) or CCP5 (bottom). The theoretical monoisotopic mass of the C-terminal fragment with no Glu side chain residues is 2632.06 (sequence AALEKDYEEVGIDSYEDEDEGEE).

FIGURE 6.

Time course of CCP1 and CCP5 activity toward β3-tubulin C-terminal peptides. Purified CCP1, CCP5, and control eluates were incubated with peptides corresponding to the C-terminal region of β3-tubulin and containing one, two, three, or four Glu residues at the side chain (sequence DATAEEEGEMYE*DDDEESEAQGPK, with the side chain glutamate(s) added to the E marked with an asterisk). After incubation for 10, 100, or 1000 min with 1 μm peptide, samples were desalted, mixed with matrix, and analyzed by MALDI MS. Top panels, peak heights representing different peptides within a spectrum were measured, and the relative abundance of each peptide was calculated as a percentage. Bottom panels, representative spectra for the peptide with four Glu residues incubated for 1000 min with control baculovirus, CCP1, or CCP5. Note that the CCP5 reaction shows more intact substrate (Peptide+4E) and also more of the fully processed product (Peptide, no E) as compared with the CCP1 reaction, indicating that CCP5 is less efficient than CCP1 at cleaving longer side chains but more active toward removing single Glu residues.