(Received for publication, January 22, 1996)
From the
After proteolytic digestion of sperm tubulin from sea urchin Paracentrotus lividus, C-terminal peptides were isolated by
chromatographic separations. The peptides were analyzed by Edman
degradation and matrix-assisted laser desorption/ionization-time of
flight mass spectrometry. About 70% of the isolated C-terminal peptides
were unmodified. The remaining modified peptides have undergone a
combination of numerous posttranslational modifications generating
significant heterogeneity of sperm tubulin. -Tubulin is modified
by detyrosylation, release of the penultimate glutamate,
polyglutamylation, and polyglycylation. Glycylation and glutamylation
can coexist within one
-tubulin isoform.
-Tubulin undergoes
polyglycylation but was not observed to be polyglutamylated. The number
of units posttranslationally added reaches 11 and 12 glycyl units on
- and
-tubulin, respectively. This is different from the
polyglycylation of axonemal tubulin in Paramecium cilia where
up to 40 added glycyl units were observed both on
- and
-tubulin.
The tubulin dimer is the structural unit of
microtubules that contribute to the maintenance of cell shape,
chromosomal segregation during mitosis, axonemal transport, and cell
motility. Both
- and
-tubulin subunits are encoded by
multigene families(1, 2) , the primary products of
which can be extensively diversified by several posttranslational
modifications. It has been reported that
-tubulin can be
acetylated on Lys-40(3) , modified by
detyrosylation/tyrosylation of the C terminus(4) , with the
possible excision of the penultimate glutamyl residue after
detyrosylation(5) . The class III
-tubulin isotype can be
phosphorylated on Ser-444 (6, 7) and
Tyr-437(8) . For both
- and
-tubulin two major
polymodifications have been recently described: polyglutamylation (9) and polyglycylation(10) . These two modifications
consist of the addition of several amino acids to the
-carboxyl
group of a glutamyl residue located in the C-terminal part of the
protein: one to at least six glutamates for
polyglutamylation(9, 11, 12, 13, 14) ,
and 3-40 glycins for polyglycylation ( (10) and (15) ). (
)Polyglutamylation contributes widely to
the high heterogeneity of mammalian brain tubulin. Polyglycylation was
originally described on axonemal tubulin from Paramecium cilia(10) . It was interesting to chemically characterize
polyglycylation on axonemal tubulin from species other than Paramecium to ascertain the occurrence of this modification.
Furthermore, since the glutamyl residues modified either by
polyglutamylation or polyglycylation are close to each other, we were
interested in determining whether these two modifications are mutually
excluded within one tubulin molecule. Preliminary immunological
studies, using GT-335 and AXO-49 monoclonal antibodies, have shown that
tubulin from sea urchin Paracentrotus lividus sperm should be
polyglutamylated (16) and polyglycylated (17) . To
answer these questions we characterized the different posttranslational
modifications occurring in the C-terminal tail of sea urchin axonemal
tubulin.
After its purification from sea urchin P. lividus spermatozoa, tubulin was digested either with endoproteinase Asp-N
or with thermolysin. The different proteolytic peptides were separated
by HPLC ()chromatography and the acidic C-terminal peptides
were characterized both by their amino acid sequence, using Edman
degradation, and by their molecular mass, using MALDI-TOF mass
spectrometry. It may be noted that the
-tubulin isotype isolated
from spermatozoa axonemes of P. lividus and identified in this
work has never been described before in this species. We determined the
different modifications localized in the C-terminal tail of axonemal
tubulin. We have observed that more than 60 and 80% of the isolated
C-terminal peptides of
- and
-tubulin, respectively, are
unmodified. Within the modified C-terminal peptides, the removal of the
residue Tyr-451 is a predominant posttranslational modification of
-tubulin. Moreover, axonemal
- and
-tubulin can be
glycylated; up to 12 glycyl residues can be added. However,
polyglutamylation was only observed on the C-terminal tail of
-tubulin, where up to six glutamyl residues can be added. It was
also observed that
-tubulin can be both glutamylated and
glycylated. From this work it appears that glutamylation and
glycylation are not mutually exclusive.
Proteolysis of
purified tubulin (1 mg) by thermolysin (Boehringer-Mannheim) was
carried out in thermolysin buffer (50 mM Tris-HCl, pH 8.0,
containing 0.1 mM CaCl) with a 1/20
enzyme/substrate ratio (w/w) for 5 h at 36 °C. Digestion was
stopped by placing the sample at -80 °C.
Figure 1: DEAE HPLC separations of acidic tubulin peptides obtained after endoproteinase Asp-N (A) or thermolysin (B) treatment. The part of the gradient where C-terminal acidic peptides are eluted is presented. Fractions 1-14, designated DEAE-1 to DEAE-14, were collected and further analyzed on a reverse phase column.
Fig. 2A shows the reverse phase HPLC analysis of the
major acidic DEAE fraction, designated DEAE-5. This chromatographic
pattern, with the elution of two major peaks, is also found for the
reverse phase separations of fractions DEAE-1 to -7. The sequence of
the peptide eluted under peak 1 of the reverse phase separation matches
with the C-terminal peptide of -tubulin beginning at position 427:
DATAEEEGEFDEEEEGDEEAA
. The mass spectrum (Fig. 2B) of this peptide reveals a molecular ion with
a mass-to-charge ratio m/z = 2324.1, which
corresponds to the unmodified
-tubulin peptide (427-447)
with one Na
adduct (MNa
calculated
average mass = 2324.1). The differences between the calculated
average mass and the experimental mass determination range from 0.1 to
1.7 Da, consistent with the accuracy of MALDI-TOF mass spectrometry in
reflect on mode. Sequence of the peptide eluted under peak 2 was
identified as the C-terminal peptide of
-tubulin beginning at
position 424:
DLAALEKDYEEVGVDSVEGEAEEEGEEY
.
The mass spectrum (Fig. 2, panel C) of this peptide
shows a molecular ion at m/z = 3105.8, which
corresponds to the unmodified
-tubulin C-terminal peptide
(424-451) (MH
calculated average mass =
3105.2).
Figure 2:
Reverse phase HPLC purification (A) of peptides eluted in fraction DEAE-5 and their mass
spectrometry analysis (B and C). Fraction DEAE-5 was
further analyzed on a C-8 column (A). Peptides purified in
this way were then characterized by their amino acid sequence and mass.
The mass spectrum of C-terminal peptide (427-447) eluted
under peak 1 and
(424-451) eluted under peak 2 are
presented in panels B and C, respectively. Average
mass-to-charge ratio m/z values of the monocharged
ion (MNa
or MH
) are indicated.
Adducts of Na
(
) and K
(
)
are observed.
Two -tubulin C-terminal peptides
isolated by reverse phase separation and analyzed both by amino acid
sequencing and mass spectrometry, beginning at amino acid Asp-424, were
found to correspond to unbranched modified peptides. The major peptide,
purified from fraction DEAE-3 gave a molecular ion at m/z = 2941.4 (MH
calculated average mass
= 2942.0) corresponding to the detyrosylated C-terminal peptide
of
-tubulin (424-450). The minor peptide, purified from
fraction DEAE-2, gave a molecular ion at m/z =
2812.4 (MH
calculated average mass = 2812.9)
corresponding to the C-terminal peptide of
-tubulin without the
penultimate glutamyl residue Glu-449 (424-449) (data not shown).
Figure 3:
Mass spectrum of polyglutamylated (A) and polyglycylated (B) C-terminal peptides of
-tubulin. Panel A presents the mass spectrum of
polyglutamylated C-terminal peptides of
-tubulin eluted under
fraction DEAE-7. Two series of molecular ions are observed. Each ion is
separated by an increment of 129 atomic mass units, the mass of one
glutamyl residue. These series, beginning with molecular ion at m/z = 3106.5 and m/z = 3589.2 correspond to the addition of glutamyl residues on
C-terminal peptide of
-tubulin tyrosylated and detyrosylated,
respectively. Panel B presents the mass spectrum of
polyglycylated C-terminal peptides of
-tubulin (424-450)
eluted under fraction DEAE-2. Two series of molecular ions are
observed. Each ion is separated by an increment of 57 atomic mass
units, mass of one glycyl residue. For each series, the m/z value of the first ion is presented. Adducts of
Na
(
) and K
(
) are
observed.
, C-terminal peptide of tyrosylated
-tubulin
(424-451),
Y, C-terminal peptide of tyrosylated
-tubulin (424-450).
Sequences of the -tubulin peptides, purified on reverse phase
column from fraction DEAE-2, were
DLAALEKDYEEVGVDSVEGEA
-. As
previously, the undetected glutamyl residue corresponding to position
445 could be posttranslationally modified. Fig. 3B presents the mass spectrum of these peptides. Again, two series of
molecular ions can be observed. In the major series, six molecular ions
are each separated by 57 atomic mass units, which is the mass of one
glycyl residue. This mass increment is typical of polyglycylated
peptides (10) . The first molecular ion of this series has an m/z = 3113.8, which corresponds to the
C-terminal peptide of detyrosylated
-tubulin (424-450) with
the addition of 3 glycyl units (MH
calculated average
mass = 3113.1). The following ions correspond to the same
peptide bearing 4-8 glycyl units. A minor series of five
molecular ions, also presenting increments of 57 atomic mass units, can
be observed. The first ion at m/z = 3527.9
corresponds to the detyrosylated
-tubulin peptide (424-450)
with the addition of one glutamyl unit and 8 glycyl units
posttranslationally added (MH
calculated average mass
= 3527.5). In this series the level of polyglycylation reaches
12 additional glycyl units. Hence, analysis of these peptides shows
that the C-terminal tail of
-tubulin can be glycylated and that
the glycyl units are posttranslationally added at least on the
-carboxylic function of the Glu-445. It was observed that
-tubulin bearing 2-8 glycyl units is always detyrosylated.
Sequences of the -tubulin peptides, purified on reverse phase
column from fraction DEAE-3, were
DLAALEKDYEEVGVDSVEGEA
-. Again, the
undetected glutamyl residue corresponding to position 445 could be
posttranslationally modified. Fig. 4shows the mass spectrum of
these C-terminal peptides of
-tubulin. In this extremely complex
mass spectrum three series of molecular ions are observed. In the major
series, six molecular ions are each separated by 57 atomic mass units.
The first molecular ion of this series has a ratio m/z = 3299.4,
which corresponds to the C-terminal peptide of detyrosylated
-tubulin (424-450), with the addition of one glutamyl unit
and 4 glycyl units (MH
calculated average mass
= 3299.3). The following molecular ions correspond to the
addition of 5-9 glycyl units. The two minor series correspond
also to the C-terminal peptide of detyrosylated
-tubulin. The
first one, beginning with the molecular ion at m/z = 3056.0, corresponds to the addition of 2-4 glycyl
units (MH
calculated average mass = 3056.1)
without the addition of a glutamyl unit. The second series, beginning
with the molecular ion at m/z = 3656.9,
corresponds to the addition of two glutamyl and 8-11 glycyl units
(MH
calculated average mass = 3656.6). When
polyglutamylation and polyglycylation were detected together, it was
observed that the addition of one glutamyl unit occurs with the
addition of 2-12 glycyl units and that addition of 2 glutamyl
units occurs with the addition of 8-10 glycyl units.
Figure 4:
Mass spectrometry analysis of C-terminal
peptides of -tubulin purified from fraction DEAE-3. Fraction
DEAE-3 was further analyzed on a C-8 column. Peptides purified in this
way were then characterized by their amino acid sequence and mass. Mass
spectrum of C-terminal peptide
(424-450) is presented.
Three molecular ion series, corresponding to glycylated peptides, are
observed. For each series, the m/z value of the first
ion is presented. Adducts of Na
(
) and
K
(
) are observed.
Characterization of peptides obtained by proteolysis of axonemal
tubulin with endoproteinase Asp-N reveals that the C-terminal tail of
-tubulin can be both polyglutamylated and polyglycylated and that
these two posttranslational modifications can occur separately or
together on one molecular species.
Figure 5:
Reverse phase HPLC purification (A) of peptides eluted in fraction DEAE-12 and mass
spectrometry analysis of fraction TL-1 (B). Fraction DEAE-12
was further analyzed on a C-8 column (A). Peptides purified by
this way were then characterized by their amino acid sequence and mass.
The mass spectrum of C-terminal peptide (430-445) eluted in
fraction TL-1 is presented in panel B. One series of three
molecular ions corresponding to glycylated peptides is observed: each
ion is separated from each other by 57 atomic mass units. Adducts of
Na
(
) and K
(
) are
observed.
By analysis of all the acidic
peptides obtained after proteolysis of tubulin with thermolysin it
appears that the C-terminal -tubulin peptides can be
polyglycylated with the addition of 1-11 glycyl units at least at
Glu-438. No other posttranslational modification was detected on the
C-terminal tail of the
-subunit.
In this study, posttranslational modifications affecting the
C-terminal tail of both - and
-tubulin purified from P.
lividus spermatozoa axonemes are analyzed and characterized. It
must be noted that the
-tubulin isotype identified in this work
has never been described before in this species (see Table 1).
The relative amount (%) of each C-terminal peptide was calculated by
dividing the amount of peptide in each reverse phase fraction
(estimated by the area of the peak detected at 214 nm) by the sum of
C-terminal peptides of
- or
-tubulin isolated. It is shown
that the majority of isolated C-terminal peptides of both
- and
-tubulin are unmodified (more than 60 and 80% respectively; see Table 2).
The C-terminal tail of -tubulin can be
polyglutamylated with the addition of 1-6 glutamyl residues and
polyglycylated with the addition of 1-8 glycyl residues. Glutamyl
and glycyl residues appear to be added at least on the
-carboxyl
function of Glu-445. Primary sequence cannot be determined beyond this
position, and thus the possibility of the existence of other sites of
modifications cannot be excluded. A minor fraction of C-terminal
peptides of
-tubulin appears to be both glutamylated and
glycylated. For these peptides at least Glu-445 appears to be a site of
modification, either for glutamylation or glycylation. Moreover the
possibility cannot be ruled out that glutamyl and glycyl residues can
be added at the same site, i.e. Glu-445, and thus within the
same lateral chain. These results show that these two posttranslational
polymodifications can coexist within one
-tubulin molecule.
Approximately 19% of the isolated C-terminal peptides of
-tubulin
are glutamylated, 4% are glycylated, and another 4% are both
glutamylated and glycylated (Table 2).
Within the unbranched
posttranslational modifications, detyrosylation was the most abundant
detected, approximately 30% of C-terminal peptides from -tubulin
being detyrosylated. It was observed that detyrosylated and tyrosylated
-tubulin are polyglutamylated almost in the same proportion (see Table 2). This is in agreement with previous results showing that
polyglutamylation and detyrosylation/tyrosyaltion are two
posttranslational modifications occurring independently on
-tubulin(19) . However, the number of added glutamyl
residues detected on detyrosylated
-tubulin is twice that of the
tyrosylated form (6 versus 3). Furthermore, polyglycylated
-tubulin with or without glutamylation was always detyrosylated. A
very small fraction of C-terminal peptides of
-tubulin were
deglutamylated by release of the Glu-450 (
2-tubulin).
For
C-terminal peptides of -tubulin the only detected
posttranslational modification was polyglycylation: 1-11 glycyl
residues can be added. At least the
-carboxyl function of Glu-438
should be one site of modification. As previously, the existence of
other sites of glycylation cannot be ruled out. Less than 20% of the
C-terminal peptides of
-tubulin are glycylated (Table 2).
Now it is important to determine if the sites of modification
described in this work are the only sites, particularly in the case of
-tubulin where the two polymodifications can occur simultaneously
on the same molecule.
The axonemal tubulin from sea urchin
spermatozoa appears to be a structure where glutamylation and
glycylation coexist and moreover can modify the same -tubulin
molecule. The possible combination of polyglutamylation with
polyglycylation leads to a dramatical increase in the molecular
complexity of the system. However, in the present work it appears that
more than 60% of axonemal tubulin of sea urchin spermatozoa is
unmodified. Thus, if each modification has a specific function, only a
few modified isoforms within a microtubular structure, such as the
axoneme, may confer specific functions to individual microtubules.
In addition to the nature of the posttranslational modifications
affecting the C-terminal tail of - and
-tubulin and their
relative proportion, it is also important to specify their distribution
along the axonemal structure. Immunofluorescence studies with specific
antibodies directed against posttranslational epitopes have been
carried out(17, 20, 21) . Furthermore, the
effect of anti-modification antibodies was tested on different motility
parameters in reactivated sea urchin spermatozoa
assays(16, 17) . The development of these
immunological studies combined with the chemical results presented in
this work could provide interesting data about the function of
posttranslational modifications of tubulin.
The two
polymodifications, glutamylation and glycylation, were originally
described in two very distinct systems: mammalian brain, where all
expressed tubulin isotypes can be polyglutamylated, and the axoneme of Paramecium cilia where all tubulin isotypes are
polyglycylated. Recently Rüdiger et al.(15) have shown that in axonemal tubulin from bull sperm,
polyglycylation was described on -tubulin, while polyglutamylation
appeared to be confined to
-tubulin. From the present study
carried out with axonemal tubulin from sea urchin it appears that these
two polymodifications can coexist on one
-tubulin molecule.