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. 2012 Nov;26(11):4468-80.
doi: 10.1096/fj.12-205047. Epub 2012 Jul 26.

A structural and functional analysis of Nna1 in Purkinje cell degeneration (pcd) mice

Hui-Yuan Wu  1 , Taiyu Wang, Leyi Li, Kristen Correia, James I Morgan
Affiliations

A structural and functional analysis of Nna1 in Purkinje cell degeneration (pcd) mice

Hui-Yuan Wu et al. FASEB J. 2012 Nov.

Abstract

The axotomy-inducible enzyme Nna1 defines a subfamily of M14 metallocarboxypeptidases, and its mutation underlies the Purkinje cell degeneration (pcd) mouse. However, the relationship among its catalytic activity, substrate specificities, and the critical processes of neurodegeneration/axon regeneration is incompletely understood. Here we used a transgenic rescue strategy targeting expression of modified forms of Nna1 to Purkinje cells in pcd mice to determine structure-activity relationships for neuronal survival and in parallel characterized the enzymatic properties of purified recombinant Nna1. The Nna1 subfamily uniquely shares conserved substrate-determining residues with aspartoacylase that, when mutated, cause Canavan disease. Homologous mutations (D1007E and R1078E) inactivate Nna1 in vivo, as does mutation of its catalytic glutamate (E1094A), which implies that metabolism of acidic substrates is essential for neuronal survival. Consistent with reports that Nna1 is a tubulin glutamylase, recombinant Nna1-but not the catalytic mutants-removes glutamate from tubulin. Recombinant Nna1 metabolizes synthetic substrates with 2 or more C-terminal glutamate (but not aspartate) residues (V(max) for 3 glutamates is ∼7-fold higher than 2 glutamates although K(M) is similar). Catalysis is not ATP/GTP dependent, and mutating the ATP/GTP binding site of Nna1 has no effect in vivo. Nna1 is a monomeric enzyme essential for neuronal survival through hydrolysis of polyglutamate-containing substrates.

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Figures

Figure 1.
Figure 1.
Construction of Nna1 and mutant Nna1 transgenic mice. A) Schematic representation of L7-Nna1 transgenes. Respective mutant Nna1 cDNA was inserted into a unique BamHI site in the fourth exon of the L7 gene. B) Comparison of mRNA expression levels in cerebellum of transgenic mice. Total RNA from cerebellum of the mouse strains depicted in Figs. 3, 4, and 9, as well as from wild-type (non-Tg, nontransgenic) and transgenic mice expressing wild-type Nna1 (Nna1) at a level that rescues Purkinje cell degeneration (8) was analyzed in parallel by RT-PCR with β-actin as control. Expression of all mutant transgenes was equivalent to or higher than the effective L7-Nna1 line. Also shown are the transgene (Tg) copy numbers for the respective strains.
Figure 2.
Figure 2.
Conservation of critical amino acids between the carboxypeptidase domains of the Nna1 family and aspartoacylase suggest acidic substrates. A) Amino acid alignment of carboxypeptidase domain of Nna1 with human ASPA and other M14 enzymes was modeled based on crystallographic data, as described previously (8, 21). Amino acids in red (H1009 and E1094) are conserved among all enzymes, those common to ASPA and Nna1 (D1007 and R1078) are in brown, and those unique to the Nna1 subfamily (F1067) are in blue. Cyan boxes highlight β sheets in the secondary structures. B) Amino acid alignment of carboxypeptidase domains of all Nna1 family members (Nna1 and CCP2-CCP6). Boxes highlight amino acids mutated in this study that are variously unique to the Nna1 subfamily (F1067Y/A) or shared with ASPA (D1007E, R1078E and E1094A). Amino acid color-coding scheme is same as panel A. Note the D1007E and R1078E mutants in Nna1 mimic Canavan disease mutations in human ASPA.
Figure 3.
Figure 3.
The catalytic site glutamate (E1094) in Nna1 is essential for activity, whereas the subfamily specific phenylalanine (F1067) in the S1′ site of Nna1 is not. A) Representative RT-PCR of total RNA from cerebellum of wild-type (non-Tg, nontransgenic) and mutant Nna1 transgenic lines. Letters indicate mutations of E1094 (Nna1E/A) and F1067 (Nna1F/Y and Nna1F/A). B) Calbindin D-28K immunohistochemistry and hematoxylin counterstaining of 2-mo-old cerebellum from wild-type (a, f), pcd3J−/− (b, g), pcd3J−/− harboring an L7-Nna1E1097A transgene (c, h), pcd3J−/− harboring an L7-Nna1F1067Y transgene (d, i), and pcd3J−/− harboring an L7-Nna1F1067A transgene (e, j) mice. Note loss of calbindin-positive Purkinje neurons in pcd3J−/− (b, g) and pcd3J−/− mice harboring an L7-Nna1E1097A transgene (c, h), whereas both the L7-Nna1F1067Y (d, i) and L7-Nna1F1067A (e, j) transgenes rescue Purkinje cell degeneration. Scale bars = 100 μm (a–e); 50 μm (f–j).
Figure 4.
Figure 4.
Two amino acids uniquely conserved between the Nna1 family and ASPA are essential for Nna1 activity. A) Representative RT-PCR of total RNA from cerebellum from wild-type (non-Tg, nontransgenic) and transgenic mice harboring mutations of D1007 (Nna1D/E) and R1078E (Nna1R/E) as well as transgenic mice expressing wild-type Nna1 (Nna1) at a level that rescues Purkinje cell degeneration. Identical mutations in ASPA homologous to D1007 and R1078 cause Canavan disease. B) Calbindin D-28K immunohistochemistry and hematoxylin counterstaining of cerebellum from 2-mo-old wild-type mice (a, e), pcd3J mice (b, f), and pcd3J/L7-Nna1D1007E (c, g) and pcd3J/L7-Nna1R1078E (d, h) transgenic mice reveals that neither mutant rescues Purkinje cell degeneration, which suggests that D1007 and R1078 are essential for activity of Nna1 in vivo and that Nna1 and ASPA have similar catalytic properties. Scale bars = 100 μm (a–d); 50 μm (e–h). C) HEK293 cells were transfected with plasmids expressing Nna1 or the indicated mutants for 40 h and then treated with CHX (50 μg/ml). Levels of Nna1 and its mutants were determined at various times following CHX addition by immunoblotting using an Nna1 antibody. Following protein synthesis, inhibition levels of wild-type Nna1 and Nna1 mutants declined at comparable rates.
Figure 5.
Figure 5.
Physical characterization of Nna1. A) SDS-PAGE reveals that purified recombinant Nna1 runs as a major band of ∼140 kDa when visualized by Coomassie brilliant blue (CBB) staining (left lane) or Western blotting (WB; middle lane). In cerebellar lysates, a band of equivalent molecular mass to recombinant Nna1 is detected in wild-type (WT) but not pcd3J mice (right panel). In both purified recombinant Nna1 preparations and cerebellar extracts from wild-type but not pcd mice, several lower-molecular-mass bands of Nna1-like immunoreactivity are detected. B) Determination of molecular mass of native Nna1 using glycerol gradient preparative ultracentrifugation. Cerebellar lysate from adult wild-type mice was subjected to centrifugation on a calibrated glycerol gradient and fractions collected and Nna1 detected by immunoblotting. Percentage (%) of glycerol (open circle) of each fraction was determined by measuring refractive index. Standard proteins (solid red diamond) are ovalbumin (43 kDa), bovine serum albumin (67 kDa), aldolase (158 kDa), catalase (232 kDa), and thyroglobulin (669 kDa). Position of standard proteins postcentrifugation in the gradient was determined by SDS-PAGE. Positions for the standard proteins were simulated with the program SEDFIT (http://www.analyticalultracentrifugation.com) and coincided with the positions determined via experimentation. The position of a 1000-kDa protein was calculated with SEDFIT, assuming a compact globular protein. Nna1-immunoactivity peaks among fractions 4–7 (lower panel), consistent with a molecular mass of ∼140 kDa. C) Determination of the molecular mass of native Nna1 using gel filtration. A cerebellum lysate was subjected to FPLC gel filtration analysis with a S200 column. Inset: calibration curve using standard proteins (red diamonds): thyroglobulin (669 kDa), ferritin (440 kDa), aldolase (158 kDa), conalbumin (75 kDa), and ovalbumin (44 kDa). Nna1-immunoactive signal appeared between fractions 21 to 26, peaking in fraction 23. Molecular mass of Nna1 (blue circle) calculated from the calibration curve is 177 kDa, suggesting that native Nna1 behaves as a monomer. Ve, elution volume; V0, void volume.
Figure 6.
Figure 6.
Nna1 but not Nna1 mutants degrade polyglutamate chain of tubulin. A) Recombinant Nna1 or heat-denatured recombinant Nna1 were incubated for the indicated times with porcine tubulin, and polyglutamylation levels were monitored by immunoblotting with the B3 antibody. Presence of Nna1 (but not heat-denatured Nna1) caused a time-dependent loss of B3-like immunoreactivity, indicating progressive degradation of polyglutamate chains. Addition of 5 mM OP inhibited the activity of Nna1. Levels of α-tubulin served as control for overall tubulin loading and stability and were not affected by Nna1. B) Incubation of tubulin with lysates from HEK293 cells transfected with an Nna1 expression plasmid also reduced B3 immunoreactivity in an OP-inhibitable fashion. C) Catalytic site mutants of Nna1 fail to decrease polyglutamylation level of tubulin. Lysates from HEK293 cells transfected with expression plasmids for Nna1 or the indicated mutants were incubated with tubulin, and B3 immunoreactivity was assessed 5 h later; whereas Nna1 reduced B3 immunoreactivity, the mutants were inactive. Levels of recombinant enzymes were monitored by immunoblotting for Nna1 and were roughly equivalent. Levels of α-tubulin served as loading and integrity control.
Figure 7.
Figure 7.
Characterization of the catalytic properties of recombinant Nna1. A) Nna1 catalyzes glutamate release from synthetic polyglutamate-containing substrates. Recombinant Nna1 was incubated with 0.5 mM FA-Glu-Glu (FA-2E), biotin-2E, or biotin-3E at 37°C for 5 h, and glutamate release was assessed using ninhydrin-Cd. Both biotin-2E and biotin-3E were much better substrates for Nna1 than FA-2E. Bars represent means ± se of triplicate determinations. B) Time courses of glutamate release from synthetic substrates. Nna1 (6 μg/ml) was incubated with either 40 μM biotin-2E (triangles) or 20 μM biotin-3E (squares) for the indicated times, and glutamate release was determined. Data are means ± se of triplicate determinations. Both substrates were metabolized in a time-dependent manner, with the initial reaction rates being higher for biotin-3E compared to biotin-2E. C) Zinc stimulates Nna1 activity. Nna1 and biotin-3E (40 μM) were incubated for 30 min with various concentrations of zinc chloride, and glutamate released was measured. Data are means ± se of triplicate determinations. Nna1 activity approximately doubled in the presence of 0.1 μM Zn2+ but was inhibited by higher concentrations. *P < 0.05. D) Nna1 is inhibited by zinc chelating agents. Nna1 was incubated with either 5 mM EDTA or OP and 40 μM biotin-3E for 30 min, and glutamate release was assessed. Both agents significantly reduced Nna1 activity. *P < 0.05.
Figure 8.
Figure 8.
Kinetic analysis of recombinant Nna1. A) Time course of Nna1 catalysis of biotin-2E and biotin-3E, assessed by mass spectrometry. Masses corresponding to biotin-1E (1E), biotin-2E (2E), and biotin-3E (3E) are indicated. A substantial fraction of biotin-3E is converted to biotin-2E within 15 min, and biotin-3E is no longer detectable by 120 min of incubation with recombinant Nna1. In contrast, conversion of biotin-2E to biotin-E is insignificant at 15 min and is only marked at 120 min, indicating that the terminal glutamate in biotin-3E is more efficiently removed than the terminal glutamate in biotin-2E. The reaction conditions were as for Fig. 5B. B, C) Lineweaver-Burk plot for Nna1 with biotin-2E (B) or biotin-3E (C) as substrates. Increasing concentrations of biotin-2E or biotin-3E were incubated with 3 μg Nna1 for 15 min, and glutamate released was measured. All reactions were performed in triplicate. Double reciprocal plots were generated, and KM and Vmax were calculated by the method of Matthews et al. (27). For biotin-2E, Vmax is 0.76 nmol/min, and KM is 0.15 mM. For biotin-3E, Vmax is 5.42 nmol/min, and KM is 0.18 mM. Binding affinity of Nna1 for substrates with 2 or 3 glutamate residues is roughly the same, but the reaction rate for removing the third glutamate is 7 times faster than removal of the second glutamate.
Figure 9.
Figure 9.
Nna1 activity is not nucleotide-dependent. A) Relative activity of recombinant Nna1 toward biotin-3E in the presence of physiological concentrations of ATP, ADP, and GTP. None of the nucleotides affected Nna1 activity at the indicated or higher concentrations (data not shown). Bars represent means ± se of triplicate determinations. B) Site of lysine to asparagine mutation (K816N) in the putative ATP/GTP binding motif of Nna1. Walker A-box type (GXXGKS) motif is underscored. C) RT-PCR of total RNA from cerebellum of wild type (non-Tg, nontransgenic) L7-Nna1K816N (Nna1K/N) and L7-Nna1 (Nna1) transgenic mice. Expression level of mRNA for L7-Nna1K816N is comparable to that in the active L7-Nna1 line. D) ATP/GTP binding motif is not essential for Nna1 activity. Calbindin D-28K immunohistochemistry and hematoxylin counterstaining in sections of cerebellum from 2 mo-old wild-type (WT; a, d), pcd3J (b, e), and pcd3J/L7-Nna1K816N mice (c, f) show preservation of Purkinje cells in the mutant transgenic line. Scale bars = 100 μm (a–c); 50 μm (d–f).
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