(Received for publication, December 31, 1996, and in revised form, February 12, 1997)
From the Department of Biological Chemistry and Mental Health Research Institute, University of Michigan, Ann Arbor, Michigan 48109
Biochemical characterization of changes in gene
expression that accompany optic nerve regeneration has led to the
identification of proteins that may play key roles in the regeneration
process. In this report, a cDNA encoding gRICH70, a novel isoform
of the regeneration-induced gRICH68 protein, has been identified and characterized in goldfish. Both gRICH68 and gRICH70 show significant homology (34-36%) to mammalian 2,3
-cyclic-nucleotide
3
-phosphodiesterases (CNPases), hence the name oldfish
egeneration-nduced NPase omolog (gRICH). The predicted 431-amino acid gRICH70
protein is 88% homologous to gRICH68, and the retinal mRNA for
gRICH70 is coordinately induced with gRICH68 mRNA during optic
nerve regeneration. Enzymatic analysis of recombinant proteins confirms
that both gRICH proteins possess CNPase activity. Despite the
relatively limited sequence homology, the kinetic constants obtained
suggest that both gRICH proteins are at least as efficient as
recombinant mouse CNP1 in catalyzing the hydrolysis of 2
,3
-cAMP.
Immunoprecipitation studies indicate that gRICH proteins are
responsible for the majority of the CNPase activity detected in
regenerating goldfish retinas. The evidence presented demonstrates that
gRICH68 and gRICH70 correspond to a previously described doublet of
acidic proteins that are selectively induced in the goldfish retina
during optic nerve regeneration. Thus, CNPase enzyme activity is
implicated for the first time in the process of nerve regeneration.
Regeneration of the axotomized optic nerve in cold blooded vertebrates has served as a model system for the characterization of cellular and molecular events underlying regeneration in the central nervous system (CNS)1 (1, 2). While warm blooded vertebrates readily regenerate peripheral nerves, the mature CNS does not support axonal regeneration. Thus, lesioned CNS axons undergo axonal degeneration and permanent loss of nervous function (3, 4). The differences involved in the loss of the regenerative capacity between the CNS and peripheral nervous system of warm blooded species on the one hand and between warm blooded and cold blooded CNS on the other are not fully understood. Experimental analysis suggests that soon after embryonic development, changes in both the neurons and the glial cell populations establish a nonpermissive environment for axonal growth in the CNS (5, 6). A better understanding of the biochemical basis of these differences will have direct relevance for the understanding and treatment of traumatic and degenerative human neurological disorders.
The goldfish visual system has been used extensively in CNS nerve regeneration studies (2, 7). After optic nerve crush, the retinal ganglion cells undergo morphological and biochemical changes (8-10) and regrow their axons to reform connections within the tectum with a high degree of spatial specificity (11, 12), resulting in recovery of visual function (13). Studies of the molecular correlates of optic nerve regeneration have implicated several proteins in the process of regrowth, including cytoskeletal components (14-16), cell adhesion molecules (17), ion channels (18), transcription factors (19), and several proteins of as yet unknown function (15, 20-25).
p68/70 is a doublet of acidic proteins that is markedly induced in goldfish retinal ganglion cells and transported into the optic nerve during optic nerve regeneration (15). The doublet was purified from goldfish tissues, and a polyclonal antibody was generated and used to demonstrate a marked increase of expression of p68/70 in the retinal ganglion cells during regeneration (26). More recently, peptide sequences obtained from the purified p68/70 (27) were used to isolate a corresponding cDNA from a goldfish nerve-regenerating retina cDNA library (28). The encoded protein was designated RICH (egeneration-nduced NPase omolog) to underscore the homology with mammalian CNPases, enzymes highly expressed in mammalian myelin with an as yet unknown biochemical function (29). The region of homology between RICH and CNPases was localized to the COOH terminus of the proteins (28), a region that contains the catalytic activity of mammalian CNPases (30). The RICH protein showed several characteristics that supported its relationship with the p68/70 doublet, including a similar isoelectric point (acidic) and amino acid composition. The RICH mRNA was found to be induced during regeneration as well (28).
It remained unclear which of the two peptides in the p68/70 doublet was encoded by gRICH. It had been established previously that the p68/70 protein components migrated anomalously in SDS-PAGE, probably due to effects of protein shape and low isoelectric point and of possible post-translational modifications (27). These characteristics probably account for the differences in apparent molecular weights of p68/70 on SDS-PAGE gels and the predicted molecular mass for gRICH68 of approximately 45 kDa (28).
In the present report, a cDNA encoding a second isoform of RICH has been isolated and sequenced. It encodes a slightly larger protein that is highly homologous to RICH. The corresponding mRNA is also induced during regeneration with a very similar time course to that for the initially characterized cDNA. The evidence reported here supports the identity of these proteins with the p68/70 doublet components, and hence, they have been renamed gRICH68 and gRICH70. Moreover, the enzymatic properties of each of the gRICH proteins have been determined, confirming that both are highly active CNPases.
Goldfish (Carassius auratus) 6-9 cm in body length were obtained from Grassy Forks Fisheries (Martinsville, IN) and maintained in aerated tanks at 25 °C. The intraorbital, unilateral optic nerve crush procedure has been described previously (13).
Library ScreeningsThe isolation of the clone g-RICH-1 from
a cDNA library from goldfish optic nerve-regenerating retina
has been described previously (28). It contained two unrelated cDNA
inserts (28). A PCR fragment containing the full ORF of gRICH68 is
described below under "Bacterial Expression and Purification of gRICH
Proteins and mCNP1." Approximately 20 ng of this fragment (template)
and the antisense primer were used to synthesize a radiolabeled probe (5 × 109 cpm/µg) by asymmetric PCR. The probe was
used to screen a goldfish genomic library (kindly provided by Dr. N. Schechter) by standard hybridization techniques (31). Clone g-RICH-G4
was purified by two further rounds of hybridization, and its insert was
mapped by restriction analysis. Southern blotting using the same probe was used to determine the restriction fragments bearing sequences of
interest. The full RICH-related cDNA insert from the g-RICH-1 clone
and a fragment from the g-RICH-G4 genomic insert (part of exon 1) were
sequenced (both strands). DNA sequencing was performed by the
dideoxynucleotide chain termination method by using a Sequenase DNA
polymerase kit (U.S. Biochemical Corp./Amersham Corp.). Sequence analysis was performed with the University of Wisconsin Genetics Computer Group and DNASTAR software packages. The sequence reported here has been submitted to the GenBank data base.
The procedure used for the RNase protection assays and the total RNA isolation from goldfish retinas in a time course experiment have been described previously (28). A 183-base pair EcoRI-BamHI fragment from the gRICH70 ORF, highly divergent from the gRICH68 corresponding sequence, was selected as template and subcloned into pBluescript-KS (Stratagene), generating plasmid pBKS-gRICH70-EB. The plasmid pBKS-gRICH70-EB was digested with EcoRI, and a 234-base-long riboprobe (109 cpm/µg) was synthesized by run-off transcription using T7 RNA polymerase (Life Technologies, Inc.) by standard protocols (31). To generate sense RNA, pBKS-gRICH70-EB was digested with BamHI, and T3 RNA polymerase (Life Technologies, Inc.) was used in the synthesis reaction. Four µg of the retina total RNAs (0, 2, 5, 9, 20, and 35 days post-optic nerve crush (PC)) were used in the sample tubes. For the sense controls, several amounts of sense RNA (0, 0.3, 1, 3, 10, and 30 pg) were mixed with 4 µg of yeast tRNA. The RNAs were hybridized to 4 × 105 cpm of antisense riboprobe. After the RNase protection assay procedure, the protected fragments (188 bases in sample tubes, 187 bases in sense controls) were separated in a 6% acrylamide sequencing gel. Radiolabeled DNA markers and 2 × 103 cpm of the antisense riboprobe were run in the gel as references. PhosphorImager (Molecular Dynamics) quantitation was performed as described previously (28).
Bacterial Expression and Purification of gRICH Proteins and mCNP1All of the enzymes used were from Life Technologies unless
otherwise indicated. The plasmid pKK233-2 (Clontech) was modified for
the purpose of expression of the gRICH proteins and mCNP1. The unique
BamHI site was removed by digestion with BamHI,
treatment with Klenow DNA polymerase, and blunt end religation. The
polycloning site was modified to include a BglII restriction
site immediately next to the ribosome binding site using a previously
described PCR mutagenesis method (32). A
BglII-PstI synthetic fragment, containing an ATG
codon encoding the initiator methionine, followed by seven codons for
histidine and by an in-frame BamHI, was inserted in the
plasmid. The insert was generated by hybridization of two oligonucleotides obtained from the University of Michigan DNA Core
Facility (5-GAT CTA CCA TGG CAC ATC ATC ACC ATC ACC ACC ACG GAT CCA
GAT CTG CA-3
and 5
-GAT CTG GAT CCG TGG TGG TGA TGG TGA TGA TGT GCC
ATG GTA-3
). The plasmid generated in this way was designated pKKR2.
Any BamHI- or BglII-flanked insert containing the
ORF of interest in frame can be cloned in the unique BamHI site of pKKR2, allowing
isopropyl-1-thio-
-D-galactopyranoside-regulated expression of an NH2 terminus heptahistidine-tagged protein
in bacteria. Specific sense and antisense primers were used to generate PCR fragments containing the full ORF of gRICH68 and gRICH70 flanked by
BglII sites, as well as the full ORF of mCNP1 flanked by
BamHI sites. A subclone containing the full cDNA insert
from clone g-RICH-8 and a subclone that fuses sequences from the
g-RICH-G4 genomic clone and the g-RICH-1 cDNA clone were used as
templates in the gRICH68 and gRICH70 PCR reactions. For the mCNP1
reaction, mouse brain total RNA was used as template in a reverse
transcriptase-PCR reaction. The fragments were subcloned in pKKR2
generating the pKKR2-gRICH68, pKKR2-gRICH70, and pKKR2-mCNP1
constructs. In all cases, the subcloned PCR fragments were fully
sequenced. No mutations were detected in the gRICH68 and gRICH70 ORF
fragments, and only silent mutations were detected in the mCNP1 ORF
fragment.
Escherichia coli (XLI-Blue strain, Stratagene) were
transformed with the pKKR2-gRICH68, pKKR2-gRICH70, and pKKR2-mCNP1
plasmids and grown at 37 °C in 0.5 liters of LB medium until they
reached an optical density (at 600 nm) of 0.3. Then
isopropyl-1-thio--D-galactopyranoside was added to reach
a concentration of 0.3-0.5 mM, and expression of the
proteins was allowed for 3 h at room temperature. The bacteria were collected by centrifugation, resuspended in 25 ml of ST-lysis buffer (10 mM Tris-HCl, pH 7.5, 100 mM NaCl, 1 mM phenylmethylsulfonyl fluoride, 1 µg/ml leupeptin, 1 µg/ml pepstatin) and lysed by sonication (total of five pulses of
1-min duration at medium intensity). The lysates were cleared of debris
by centrifugation at 10,000 × g for 10 min. Imidazole
(pH 7) was added to reach a final concentration of 20 mM,
and the lysates were loaded on a nickel-nitrilotriacetic acid column
(0.5 ml of resin, Quiagen). The column was washed with ST-20 buffer (10 mM Tris-HCl, pH 7.5, 100 mM NaCl, 20 mM imidazole) and, if necessary, with ST-50 buffer (same as
ST-20 but with 50 mM imidazole). The affinity-purified
proteins were eluted with ST-250 buffer (same as ST-20 but with 250 mM imidazole). Glycerol was added to reach a final
concentration of 20%, and the purified proteins were stored at
80 °C. A working dilution of the proteins (50 ng/µl) was stored
at
20 °C in ST buffer containing 50% glycerol with no apparent
loss of enzyme activity for several months. Protein concentrations were
determined (Bio-Rad) on a Beckman Biomek-1000 automated workstation.
SDS-PAGE was used to monitor the purification procedure and the
purified proteins (Bio-Rad minigel apparatus). The purified
histidine-tagged proteins (total tag size 12 amino acids) were
designated H7-gRICH68, H7-gRICH70, and H7-mCNP1.
CNPase activity was
measured with an alkaline phosphatase (AP)-coupled assay method based
on a previously published procedure (33). The reactions were performed
in CNPase reaction buffer (50 mM MES, pH 6) at 30 °C for
20 min in a final volume of 100 µl containing the indicated
concentrations of 2,3
-cAMP as substrate (Sigma). Five ng of
H7-gRICH68, 2.5 ng of H7-gRICH70, and 10 ng of H7-mCNP1 were used in
the assays. The CNPase reaction was stopped by boiling for 1 min, and
the coupled reaction was initiated then by the addition of 50 µl of
CAP-buffer (300 mM Tris-HCl, pH 9, 21 mM
MgCl2) containing 1 unit of calf intestine AP (Boehringer) and was incubated at 37 °C for 20 min. The released inorganic phosphate was detected by a sensitive chromogenic method (34). All
measurements were performed in triplicate and were corrected with
control values obtained in parallel in assays containing all of the
components but the specific CNPase enzyme. The measured rates were used
to obtain Lineweaver-Burk plots using SigmaPlot software (Jandel).
CNPase reactions were performed with 10 mM
2,3
-cAMP in CNPase reaction buffer for 30 min at 30 °C in a final
volume of 50 µl. One µg each of H7-gRICH68, H7-gRICH70, and
H7-mCNP1 were used. The products of the reactions were separated by
paper chromatography as described previously (35). Five µl of each of
the reactions were loaded side by side with 5 µl each of a 10 mM solution of 2
,3
-cAMP, 2
-AMP, or 3
-AMP, employed as
standards for comparison (all from Sigma). The developed chromatogram
was photographed under short wave ultraviolet light
transillumination.
The PCR
fragments containing the ORFs for mCNP1, gRICH68, and gRICH70
(described above under "Bacterial Expression and Purification of
gRICH Proteins and mCNP1") were subcloned into the unique
BglII site of pCMVneo (36), generating the plasmids
pCMVneo-mCNP1, pCMVneo-gRICH68, and pCMVneo-gRICH70. Thirty µg of
each of these constructs and of the parental pCMVneo vector were
transiently transfected into HEK-293 cells (100-mm Petri dishes at 50%
confluency) by the calcium phosphate coprecipitation method (31).
Forty-eight h after transfection, the medium was removed, and the plate
was stored at 80 °C for future lysate preparation.
The transfected HEK-293 cells and the goldfish retinas
(nerve-regenerating and control, 35 days PC) were resuspended,
respectively, in 250 and 500 µl of STT lysis buffer (10 mM Tris-HCl, pH 7.5, 100 mM NaCl, 0.5% Triton
X-100, 1 mM phenylmethylsulfonyl fluoride, 1 µg/ml
leupeptin, 1 µg/ml pepstatin) and Dounce-homogenized. Glycerol was
added to a final concentration of 20%, and the lysates were stored in
aliquots at 80 °C for further analysis. Protein concentrations of
the extracts were determined as described above (under "Bacterial
Expression and Purification of gRICH Proteins and mCNP1"). The
protein extracts obtained were designated HEK-neo, HEK-mCNP1,
HEK-gRICH68, HEK-gRICH70, gf-retina-R35 and gf-retina-C35.
A polyclonal antibody was generated against gRICH protein by injection of purified H7-gRICH68 into rabbits (Research Genetics). The anti-gRICH antiserum, collected 10 weeks after the initial injection of the antigen (with two additional boost immunizations), contained a high titer of anti-gRICH antibodies as detected by Western blotting and enzyme-linked immunosorbent assay with the purified gRICH proteins. Only slight cross-reactivity with mCNP1 could be detected, even with larger amounts of mCNP1. Western blotting was used to analyze the expression in transfected HEK-293 and goldfish retina extracts. Two µg of total protein from the HEK-neo, HEK-mCNP1, HEK-gRICH68, and HEK-gRICH70 extracts were loaded side by side with 40 µg of total protein from the gf-retina-R35 and gf-retina-C35 extracts in 10% SDS-PAGE. Fifty ng of the purified histidine-tagged proteins were also loaded as controls. A 1:1000 dilution of anti-gRICH antiserum was used as primary antibody. A 1:5000 dilution of goat anti-rabbit-AP (Life Technologies) was used as secondary antibody. The blots were developed with the nitro blue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate substrate system (Life Technologies, Inc.). Additionally, the blots were photographed with a Kodak DC40 digital camera for quantitation with Kodak 1D image analysis software.
CNPase assays were performed with the extracts by the AP method
(described under "Determinations of Kinetic Constants"). The concentration of the substrate, 2,3
-cAMP, was 4 mM. The
amounts of total protein used as a source of enzyme were 0.1 µg of
the HEK-neo, HEK-mCNP1, HEK-gRICH68, and HEK-gRICH70 extracts and 1 µg of the gf-retina-R35 and gf-retina-C35 extracts. Assays were performed in triplicate, and control values were obtained from assays
with all of the components except the enzyme source and subtracted from
the values obtained with the extracts.
The HEK-mCNP1, HEK-gRICH68, and HEK-gRICH70 extracts were used as positive controls. They were diluted to 0.05 µg/µl in ST-0.1% T buffer (10 mM Tris-HCl, pH 7.5, 100 mM NaCl, 0.1% Triton X-100), and 50 µl of the diluted extracts were then incubated with 5 µl of anti-gRICH antiserum at 4 °C for 1 h in a horizontal rotator. Next, 50 µl of a suspension of protein A-Sepharose beads (Pharmacia Biotech Inc.) in ST-0.1% T buffer (40% beads) was added and incubated at 4 °C for 1 h in a horizontal rotator. The tubes were then spun in a microcentrifuge, and the immunodepleted supernatants were transferred to new tubes. For the gf-retina-R35 extract, the same procedure was followed except that the extract was diluted to 1 µg/µl and the volumes used were half of those described for the controls. In all cases, control immunodepletions were performed with the preimmune serum obtained from the same rabbit. All of the immunodepletion procedures were performed in triplicate. The immunodepleted supernatants were used in CNPase assays by the AP method (described under "Determinations of Kinetic Constants"). The determined specific activities were then normalized to the results obtained with the preimmune controls for the same extract.
The isolation of
a cDNA encoding RICH, based on peptide sequences derived from
purified p68/70 has been previously reported (28). A third clone
(g-RICH-1) was obtained in the original screening, but the cDNA
insert of that clone was not fully characterized at the time, since it
did not contain a full ORF and was in fact the result of the fusion of
two unrelated cDNAs (28). Therefore, a goldfish genomic library was
screened to obtain the sequences corresponding to the 5
-end of the new
mRNA that were missing in the g-RICH-1 cDNA insert. Clone
g-RICH-G4 was isolated in this screening, and partial sequence of the
region immediately upstream of that of the g-RICH-1 cDNA insert was
determined. The sequence obtained showed continuous homology to that of
the g-RICH-8 cDNA up to the 5
-untranslated region, 25 bases
upstream of the initiator ATG codon, suggesting that this sequence
corresponded to a single exon. The composite sequence is represented in
Fig. 1. Evidence presented below has indicated the
identity of the two isoforms of RICH with the components of the p68/70
protein doublet. The first isoform (28) corresponds to the faster
migrating protein of the p68/70 doublet, while the novel isoform
described here corresponds to the slower migrating protein; hence, they
have been renamed gRICH68 and gRICH70, respectively. The predicted gRICH70 protein is 431 amino acids in length, 20 amino acids longer than gRICH68, and has a predicted molecular mass of 46,972 Da and a
calculated isoelectric point of 4.39.
An alignment of gRICH70 with gRICH68 is presented in Fig.
2. The two isoforms are highly homologous (88% amino
acid identity) over their entire sequence, and both proteins contain an
identical isoprenylation consensus sequence (CTIL) at the COOH terminus (37). The homology with mammalian CNPases is clearly localized to the
COOH terminus (Fig. 2), corresponding to the catalytically active
region of these proteins (30).
gRICH70 mRNA Is Induced in Nerve-regenerating Retinas during Goldfish Optic Nerve Regeneration
A significant induction of the
gRICH68 mRNA during goldfish optic nerve regeneration has been
previously demonstrated by a specific RNase protection assay (28). In
similar studies of the gRICH70 mRNA, induction was detected in the
regenerating retinas (R lanes) as early as 2 days PC and
continued to increase up to 20 days PC, declining at later times (Fig.
3A). No significant induction was detected in
the control retinas (C lanes). This time course of the
induction resembled that seen previously for the gRICH68 mRNA,
suggesting coordinate regulation of gRICH68 and gRICH70 mRNAs.
Sense RNA controls were included in the assay for accurate quantitation
in a PhosphorImager. The quantitation indicated a maximal induction
of gRICH70 mRNA of 6-fold over control retina levels 20 days PC
(Fig. 3B). It also suggested that the levels of gRICH70
mRNA are approximately 2-3-fold lower than those of gRICH68 in
normal retinas. These retinal RNA samples have been previously tested
by an RNase protection assay with a goldfish 18 S rRNA riboprobe and
yielded a band of similar intensity in all of the lanes (28).
The induction detected with whole retinas is likely to be an underestimation of the induction in the retinal ganglion cell layer, since constitutive expression of p68/70 proteins has been demonstrated in other retinal layers (26).
Bacterial Expression and Purification of gRICH Proteins and mCNP1The predicted gRICH proteins showed very significant
homology to the mammalian myelin CNPases, particularly in the catalytic region of these proteins, including an isoprenylation motif at the COOH
terminus (Fig. 2 and Ref. 28). CNPases hydrolyze
2,3
-cyclic-nucleotide monophosphates specifically to the
2
-nucleotide monophosphate product, but their in vivo role
is as yet unclear (29).
To explore further the significance of the homology between the gRICH
proteins and mammalian CNPases, gRICH68 and gRICH70 proteins were
expressed and purified from E. coli. mCNP1 was also purified
using the same system. Bacterial expression constructs were designed so
that the full ORFs for the three proteins were fused in frame to a
sequence encoding a heptahistidine tag. The NH2-terminal
heptahistidine-tagged proteins (H7-gRICH68, H7-gRICH70, and H7-mCNP1)
could therefore be purified from the bacteria to near homogeneity by a
single column affinity purification method (described under
"Materials and Methods"). Five µg of each of the purified
proteins were analyzed by SDS-PAGE (Fig. 4). While the
three proteins (mCNP1, gRICH68, and gRICH70) are of similar length
(400, 411, and 431 amino acids long, respectively), their apparent
molecular weight in SDS-PAGE is significantly different. While mCNP1
seems to migrate accordingly with the predicted molecular weight, gRICH
proteins migrate significantly more slowly, slightly below the 71 kDa
molecular mass standard (Fig. 4). These electrophoretic mobilities
support the assignment of gRICH proteins with the p68/70 doublet. The
anomalous migrations may be dependent on the sequence of the proteins
themselves, since the relevant post-translational modifications do not
occur in bacteria.
gRICH Proteins Are Novel and Efficient CNPases
The purified
H7-gRICH68 and H7-gRICH70 were tested for CNPase activity. Assays with
H7-mCNP1 were performed for comparison. Initial rates of reaction were
determined in assays performed in triplicate at several concentrations
(0.25-8 mM) of the substrate 2,3
-cAMP, and the values
were used to obtain Lineweaver-Burk plots. A representative experiment
is presented in Fig. 5. Vmax and
Km values obtained from three independent
experiments for each enzyme were then averaged (Table
I). The kinetic constants obtained for the recombinant
gRICH proteins indicated that they are more efficient CNPases than the
recombinant mCNP1 (higher Vmax and lower
Km).
|
The results determined with recombinant mCNP1 cannot be directly compared with the kinetic constants obtained with native proteins purified from myelin, since mammalian CNPases have been purified only from other species and have been tested under different conditions (29). However, the Km values for all three (H7-mCNP1, H7-gRICH68, and H7-gRICH70) fall well within the range determined for mammalian CNPases purified from myelin. The Vmax values are slightly lower than those reported with highly purified mammalian CNPases (approximately 800-1000 units/mg) (29). In other experiments, a different bacterial expression system (New England Biolabs), which generates proteins fused to the E. coli maltose-binding protein (MBP), was used to purify MBP-mCNP1 and MBP-gRICH68. Both enzymes were active and yielded virtually identical kinetic constants to those obtained with the corresponding heptahistidine-tagged proteins (data not shown).
The CNPase activity assay employed does not distinguish the product
isomer (2-NMP or 3
-NMP) generated in the reaction. Mammalian CNPases
hydrolyze 2
,3
-cNMP substrates exclusively to the 2
-NMP, while RNases
most often yield the 3
-NMP. It was therefore of interest to analyze
the product generated from the hydrolysis of 2
,3
-cAMP by gRICH
proteins. H7-gRICH68 and H7-gRICH70 generated the 2
-AMP specifically
as did H7-mCNP1, confirming that these proteins are true CNPases (Fig.
6).
gRICH Proteins Are Equivalent to the p68/70 Doublet Components
To compare directly the gRICH68 and gRICH70 proteins encoded in the cDNAs with the p68/70 doublet from goldfish, a polyclonal antibody recognizing specifically gRICH proteins was generated. Purified H7-gRICH68 was used as the antigen for rabbit immunizations (see "Materials and Methods"), and the antibody was characterized using the purified proteins. The anti-gRICH antibody recognized both H7-gRICH68 and H7-gRICH70 and showed very weak cross-reactivity with H7-mCNP1.
Constructs for the eukaryotic expression of the encoded gRICH68 and gRICH70 proteins were generated and transfected transiently into HEK-293 cells, a system that can produce isoprenylated proteins. Control transfections were performed with the parental plasmid (pCMVneo) and with a construct expressing mCNP1 as well. Protein extracts were obtained from the transfected HEK-293 cells (HEK-neo, HEK-mCNP1, HEK-gRICH68, and HEK-gRICH70 extracts) as well as from goldfish nerve-regenerating and control retinas 35 days PC (gf-retina-R35 and gf-retina-C35 extracts).
The protein extracts were analyzed by Western blot (Fig.
7). The anti-gRICH antibody detected a doublet of
proteins in the 68-70-kDa area in the goldfish extracts. Additionally,
the doublet was induced in the optic nerve-regenerating retinas and
comigrated perfectly with the major protein products detected in the
HEK-gRICH68 and HEK-gRICH70 extracts. All these data indicate that
gRICH68 and gRICH70 proteins are identical to the p68/70 doublet
components. Quantitation of the Western blot signal demonstrated an
induction of the gRICH68/gRICH70 proteins detected in the regenerating
retinas of 4.17 ± 1.40 fold, a result correlating with the
induction detected previously for the p68/70 doublet itself (26). No
specific signal was detected in the HEK-neo or HEK-mCNP1 extracts (Fig.
7).
Analysis of the Enzymatic Activity of Recombinant and Native gRICH Proteins
The protein extracts used for the Western blot analysis
were also tested for CNPase activity by the previously described
AP-coupled method. A representative experiment is presented in Fig.
8. As expected, the HEK-mCNP1, HEK-gRICH68, and
HEK-gRICH70 extracts showed high specific activity compared with the
control HEK-neo extract (Fig. 8A). The activity detected in
the HEK-mCNP1 extract confirmed the presence of the enzyme, indicating
that the absence of a band in the Western blot (Fig. 7) was not due to
lack of expression but to the specificity of the anti-gRICH antibody. An estimation of the Vmax of the eukaryotic
recombinant gRICH68 and gRICH70 proteins can be calculated based in the
levels quantitated in the Western blot (Fig. 7; assuming that
anti-gRICH recognizes equally the bacterial and eukaryotic proteins)
and the specific activity levels of the extracts. This calculation
yields values in the range of 500-1000 µmol min1
mg
1, correlating fairly well with the values obtained
with the purified bacterial proteins, suggesting that the
post-translational modifications in eukaryotic cells do not
significantly affect the catalytic properties of the enzymes.
The activity levels in goldfish retina were much lower than those in the overexpressing extracts (Fig. 8A), correlating well with the lower levels of gRICH proteins detected in the Western blot (Fig. 7). However, an induction of CNPase activity could be clearly detected in the optic nerve-regenerating retinas over the levels of control retinas (Fig. 8B). The average induction of three independent experiments was 3.54 ± 0.84-fold. The induction of the CNPase activity correlates well with the induction of the gRICH doublet detected by Western blot (Fig. 7). This result further supports the identity of gRICH68 and gRICH70 with the p68/70 doublet components. Moreover, it suggested that the gRICH proteins are contributing significantly to the total CNPase activity in retinas.
The Majority of the CNPase Activity in Nerve-regenerating Retinas Is Due to gRICH ProteinsFrom the analysis of the retina
extracts, it could be inferred that a significant fraction of the
CNPase activity detected was due to the gRICH proteins. To directly
explore the proportion of the CNPase activity attributable to gRICH
proteins, immunodepletion experiments with the anti-gRICH antiserum
were performed. The gRICH proteins present in the extracts were
immunoprecipitated by binding to the anti-gRICH polyclonal antibody,
followed by precipitation with protein A-Sepharose beads, and the
immunodepleted supernatants were tested for CNPase activity. As
expected, the CNPase activity was severely reduced in the HEK-gRICH68
and HEK-gRICH70 extracts by the immunodepletion procedure, while CNPase
activity was not significantly affected in the HEK-mCNP1 extract (Fig. 9). Further controls indicated that the preimmune serum
did not have a significant effect on the activity of any extract. In
addition, the anti-gRICH antibody per se (without the
protein A-Sepharose precipitation step) was able to block the activity
of the gRICH enzymes only by approximately 30-40% (data not shown).
The immunodepletion protocol removed most of the CNPase activity from
the gf-retina-R35 extract (>98%; Fig. 9). This result was confirmed
by three independent experiments and provides direct evidence
indicating that the majority of the CNPase activity in
nerve-regenerating retinas is accounted for by the gRICH68 and gRICH70
proteins.
In this report, previous biochemical studies of optic nerve regeneration in goldfish (7, 26-28) have been extended with the cloning of a cDNA encoding a novel isoform of RICH proteins. The available cDNAs have allowed the expression of the encoded proteins both in prokaryotic and eukaryotic recombinant systems, as well as the generation of a specific polyclonal antibody that recognizes the gRICH proteins efficiently. The experimental evidence obtained confirmed the identity of gRICH68 and gRICH70 with the p68/70 protein doublet components, two induced proteins previously identified in the goldfish optic nerve regeneration model system.
The studies performed here with the purified recombinant proteins have identified gRICH68 and gRICH70 as efficient CNPases. These novel CNPases are the first cloned nonmammalian members of this family of enzymes. Since they are nevertheless related to the mammalian CNPases, they should be helpful in delimiting the regions of the protein relevant for the enzymatic activity. These highly conserved motifs will probably correspond to those relevant for the in vivo function of these proteins. Of note, those conserved sequences do not include several sequence motifs that were previously thought to be involved in catalysis (28, 29).
The gRICH68 and gRICH70 proteins showed very similar migrations in SDS-PAGE and enzymatic activity when expressed in E. coli or HEK-293 cells, suggesting that post-translational modification is not the cause of the anomalous migration of gRICH proteins, nor is it necessary for enzymatic activity. Alternatively, the acid pI of both gRICH68 and gRICH70 relative to mCNP1 may be responsible for the anomalous migration in SDS-PAGE. The overexpression systems described here should be useful for the studies of post-translational modifications of gRICH proteins and to discern their role on the gRICH proteins function. The isoprenylation of mammalian CNPases have been shown to be important for their membrane localization and for the phenotypic effects of their overexpression (38, 39).
The two known isoforms of mammalian CNPases, CNP1 and CNP2, differ only
in the amino terminus. They arise by alternative splicing and promoter
usage (40, 41). The gRICH68 and gRICH70 cDNAs showed differences
all along the sequence, suggesting that they are derived from two
distinct genes. Both the gRICH68 and gRICH70 mRNAs showed similar
time courses of induction during optic nerve regeneration (Ref. 28 and
Fig. 3). The mechanisms of transcriptional induction of specific genes
following nerve injury are not well understood. The time course of
induction of gRICH mRNAs closely resembles that of the -tubulin
(18), with a progressive increase in message levels during the period
of axonal regrowth. The levels of a nicotinic acetylcholine receptor
subunit mRNA follow a different time course, being induced late in
the regeneration process (18), corresponding to the period of synapse
reformation. The study and comparison of the promoters of the induced
genes should lead to a better understanding of the signaling mechanisms
underlying the transcriptional control during this process.
The expression systems utilized in these studies demonstrated that gRICH68 and gRICH70 are novel members of the CNPase family of enzymes. The best studied members of this family are the mammalian CNPases (29), although enzymes with CNPase activities have been detected in organisms as diverse as crustaceans (42) and even plants (43). The mammalian CNPases are very highly expressed in oligodendrocytes and Schwann cells, and they have been widely used as a marker of these glial cells (29). Additionally, CNPases are strongly associated with myelin membranes both by a hydrophobic isoprenyl group in their COOH terminus (38, 39) and by interaction with the cytoskeleton (44, 45). Overexpression experiments in cell lines (39) and in transgenic mice (46) have suggested that they are involved in the generation of membranous extensions and myelin biogenesis. The conservation between gRICH proteins and mammalian CNPases of both the enzymatic activity and the COOH-terminal isoprenylation signals strongly suggests that they must have related biochemical roles in vivo. This is particularly curious inasmuch as it is clear that, in the goldfish visual system, gRICH proteins are induced in neurons (the retinal ganglion cells) and are axonally transported down the regenerating nerve fibers (26). A possible explanation for the appearance of CNPase activity in neurons may be found in a functional commonality between the glial cell that is proliferating large amounts of membrane and supporting cytoskeleton (myelin) and the regenerating cell body of neurons, which are engaged similarly in the generation of long axonal processes. In each instance, the cell supports a massive outgrowth at a great distance from the cell body and nucleus. Although CNPase-immunoreactive protein has previously been identified in neuronal tissue (47), the present studies implicate this activity in nerve regeneration for the first time. Further studies may lead to a better understanding of the molecular basis of nerve regeneration as well as to the identification of the true physiological substrate of CNPases.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) U82989[GenBank].
We thank Marianne R. Lewis for technical assistance.