(Received for publication, November 10, 1994)
From the
The heavy chain of human glycosylasparaginase (N-(
-N-acetylglucosaminyl)-L-asparaginase
(EC 3.5.1.26)) has five cysteinyl residues (Cys-61, Cys-64, Cys-69,
Cys-163, and Cys-179). A Cys-163 to serine substitution due to a point
mutation in the glycosylasparaginase gene causes the most common
disorder of glycoprotein degradation, the Finnish-type
aspartylglycosaminuria. To localize the potential disulfide bonds, the
isolated heavy chain of human leukocyte glycosylasparaginase was
treated with the enzyme
-chymotrypsin, and the resulting peptides
were separated by high performance liquid chromatography prior to and
after reduction and S-carboxymethylation. The peptide
containing the Cys-163 residue and the peptide to which it is connected
with a disulfide were structurally characterized by mass spectrometry.
The disulfide bond crucial for catalytic activity, subunit processing,
and biological transport of glycosylasparaginase was located close to
the carboxyl terminus of the heavy chain at positions 163 and 179.
Glycosylasparaginase (glycoasparaginase, N-(
-N-acetylglucosaminyl)-L-asparaginase
(EC 3.5.1.26)) is a lysosomal enzyme that cleaves the linkage between N-acetylglucosamine and L-asparagine causing
degradation of N-glycosidic glycopeptides. The structure,
enzymatic characteristics, mechanism of action, and substrate
specificity of human leukocyte glycosylasparaginase have been recently
described(1, 2) . Glycosylasparaginase is encoded as
an enzymatically inactive precursor polypeptide that is
post-translationally processed to two subunits, 25-kDa heavy and 19-kDa
light chains, which are connected by noncovalent forces(3) .
The heavy chain has 5 cysteinyl residues (Cys-61, Cys-64, Cys-69,
Cys-163, and Cys-179), and the light chain has 4 (Cys-286, Cys-306,
Cys-317, and Cys-345), respectively. Two single-base changes in both
alleles of the glycosylasparaginase gene, resulting in the replacement
of arginine by glutamine (Arg-161
Gln) and cysteine by serine
(Cys-163
Ser) on the heavy chain of the enzyme are found in 95%
of the Finnish AGU (
)patients(4, 5, 6) . The Cys-163
Ser substitution is known to destroy the catalytic activity,
subunit processing, and lysosomal targeting of glycosylasparaginase (5) . This cleavage is responsible for activation of the enzyme (7) by creating the amino-terminal residue of the light chain,
Thr-206, which is involved in the mechanism of enzyme action, (1) and is highly conserved among species(8) .
Aspartylglycosaminuria (McKusick 20840) is an autosomal recessive
lysosomal storage disease caused by a deficiency in
glycosylasparaginase. It is characterized by serious psychomotor
retardation and accumulation of large amounts of glycoasparagines in
body fluids and tissues(8) . AGU is the most common disorder of
glycoprotein degradation and its frequency in the Finnish population
(1:4000) is as high as that of the most commonly inherited lysosomal
storage diseases (Tay-Sachs disease and Gaucher disease type I) among
Ashkenazi Jews. In this paper we describe the localization of the
disulfide bond in glycosylasparaginase, which is disrupted by the
Cys-163 Ser mutation causing the Finnish-type AGU.
Glycosylasparaginase was isolated from human leukocytes and
purified 4600-fold(1) . The enzyme had a specific activity of
2.2 units/mg of protein. The light (19 kDa) and heavy (25 kDa) subunits
of the native 88-kDa enzyme were isolated by reversed-phase HPLC.
Peptides were produced from the heavy chain by treatment of the protein
overnight with -chymotrypsin in 50 mM ammonium
bicarbonate, pH 8.6, containing 2 M urea and 5 mM iodoacetic acid. An aliquot of the digested material was analyzed
by C
reversed-phase HPLC using a fused silica micro-column
(198-µm inner diameter) and electrospray ionization mass
spectrometry (LC-MS). A second aliquot was removed from the digested
material, reduced for 1 h with dithiothreitol, and treated with
iodoacetic acid. This aliquot was then analyzed by LC-MS.
Electrospray ionization mass spectrometry and tandem mass
spectrometry were performed as described
previously(9, 10) . The electrospray ionization tandem
mass spectrometer, TSQ-700, was obtained from Finnigan MAT (San Jose,
CA) and equipped with an Analytica ESI source (Branford, CT). The ABI
140B dual syringe pump (Applied Biosystems, Foster City, CA) was used
for HPLC, and a sample injector with a 5-µl loop was obtained from
Rheodyne (Cotati, CA). Peptides were eluted with a linear gradient of
5-90% Solvent B (80:20 acetonitrile, 0.5% acetic acid) over 30
min. The flow rate from the pumps was 100 µl/min. The solvent
stream was split, 50:1-100:1, precolumn, and the final flow
rate was 1-2 µl/min. Electrospray conditions were as follows:
sheath gas, nitrogen at 0.2 liters/min; drying gas, heated nitrogen
(80-85 °C, 16 psi) 4 liters/min; needle distance to
capillary, 2.5 cm; capillary voltage, -3500-4100 V.
A
1-µl aliquot of sample was injected to record the molecular weight
of the peptides produced by -chymotrypsin digestion. Spectra were
acquired by scanning the first mass analyzer or the second mass
analyzer at a rate of 500 u/s over the range 400-1500 m/z. Sequence analysis of peptides was performed
during a second HPLC analysis using tandem mass spectrometry with an
equivalent amount of material. The peptide of interest was isolated by
selecting the precursor ion with a 3-4-u (full width half height)
wide window in the first mass analyzer and passing the ions into a
collision cell filled with argon to a pressure of 3-5 millitorr.
Collision energies (laboratory frame) were on the order of 20-50
eV. The fragment ions produced in the collision cell were transmitted
to the second mass analyzer, which was scanned at 500 u/s over a mass
range of 50 u to the molecular weight of the precursor ion to record
the fragment ions. The electron multiplier setting was 200-400 V
higher than used to record the ions in main beam. The second mass
analyzer was tuned to give peaks which were 1.5 u wide.
The amino acid sequence of human glycosylasparaginase heavy
chain and the presumed cleavage sites produced by using
-chymotrypsin are shown in Fig. 1. The nonreduced protein
was treated with the enzyme
-chymotrypsin in the presence of 5
mM iodoacetic acid, and the resulting mixture of peptides was
analyzed by LC-MS. The total ion chromatogram is shown in Fig. 2A. An aliquot of the digested protein was reduced
with dithiothreitol and S-carboxymethylated with iodoacetic
acid. This mixture of peptides was analyzed by LC-MS (Fig. 2B), and an increase in the number of peptides
was observed. A comparison of the m/z values of
peptides expected upon treatment of the protein with
-chymotrypsin
and those observed in the reduced and S-carboxymethylated
mixture of peptides produced an ion that corresponds to the amino acid
sequence LARNCQPNYW ((M + 2H)
, m/z 662) containing the Cys-163 residue mutated in AGU. Tandem mass
spectrometry analysis of this ion confirmed the sequence assignment (Fig. 3). Calculation of the masses for all possible disulfide
configurations with this peptide, and examination of the m/z values for the peptides observed in the LC-MS
analysis of the nonreduced peptides, identified only one possibility.
An ion of m/z 851 ((M + 2H)
)
corresponds to a peptide containing the sequences LARNCQPNYW and CGPY.
This ion was not observed to an appreciable extent in the peptide
mixture produced upon reduction and S-carboxymethylation.
Tandem mass spectrometry analysis produced sufficient sequence
information to confirm the assignment of LARNCQPNYW and CGPY as
disulfide bridged peptides (Fig. 4). Thus, Cys-163 and Cys-179
are determined to be covalently bonded, and this bond is disrupted by
mutation of Cys-163
Ser in the Finnish-type
AGU(4, 5) . The present finding demonstrates on the
protein level the cause of glycosylasparaginase deficiency in over 95%
of cases of the most common disorder of glycoprotein degradation, the
Finnish-type aspartylglycosaminuria. The presence of another potential
disulfide bond between Cys-61, Cys-64, or Cys-69 could not be resolved
since the residues are located close to one another and an
-chymotryptic cleavage site doesn't exist between the
residues (Fig. 1). No other commercially available enzymes were
capable of cleaving the amino acid residues necessary to determine the
presence of additional disulfide bonds.
Figure 1:
Amino acid sequence data on human
glycosylasparaginase and localization of the disulfide bond disrupted
by a mutation in the Finnish-type aspartylglycosaminuria. The amino
acid residues of the heavy chain of human glycosylasparaginase are
numbered as in (4) . The 5 cysteinyl residues are indicated in circles, and the presumed -chymotrypsin cleavage sites
are indicated in squares.
Figure 2:
HPLC chromatogram of the heavy chain after
digestion with -chymotrypsin prior (A) and after (B) reduction and
S-carboxymethylation. A, the peak marked L1 was shown
to represent the peptide containing the disulfide bond between Cys-163
and Cys-179 (m/z, 851). B, the appearance of
the S-carboxymethylated peptide containing Cys-163 is marked
by L2 (m/z 662).
Figure 3:
Tandem mass spectrum of the peptide
LARNCQPNYW. Collision induced dissociation mass spectrum recorded on
the (M + 2H) ions at m/z 662.
Fragments of type b and y, ions having the general formulas
H(NHCHRCO)
and
H
(NHCHRCO)
OH
,
respectively, are shown above and below the deduced amino acid sequence
at the top of the figure. Ions observed in the spectrum are underlined. Leu and Ile were assigned by correspondence to the
nucleotide derived sequence. The spectrum was acquired using a
collision energy of approximately 22 eV and a gas pressure of 3.5
millitorr. The second mass analyzer was scanned at a rate of 500
u/s.
Figure 4:
Tandem
mass spectrum of the peptide disulfide bonded peptides LARNCQPNYW and
CGPY. Collision-induced dissociation mass spectrum recorded on the (M
+ 2H) ions at m/z 851.
Fragments of type b and y, ions having the general formulas
H(NHCHRCO)
and
H
(NHCHRCO)
OH
,
respectively, are shown above and below the deduced
amino acid sequence at the top of the figure. Ions observed in
the spectrum are underlined. Leu and Ile were assigned by
correspondence to the nucleotide derived sequence. The spectrum was
acquired using a collision energy of approximately 10 eV and a gas
pressure of 3.5 millitorr. The second mass analyzer was scanned at a
rate of 500 u/s.
The presence of a disulfide bond between Cys-163 and Cys-179 suggests that stabilization or correct folding of the carboxyl terminus of the heavy chain of glycosylasparaginase by a disulfide bridge 26 residues prior to the cleavage site and Thr-206 (1, 7) is required for activation of the enzyme. The Thr-206 residue at the cleavage site is preceded by 11 hydrophilic residues at the carboxyl terminus of the heavy chain. These residues have been proposed to make the region susceptible to proteolytic hydrolysis in the lysosomes(3) . The Thr-206 residue is located at the amino terminus of the newly formed light subunit in a protein sequence that is highly conserved among species (8) and that is located at or close to the active site of glycosylasparaginase(1) . Stabilization and/or proper folding of the protein chain by a disulfide bond preceding the processing site may be of more general importance in studies for the processing mechanism for this enzyme, which is currently poorly understood. Whether it involves an actual processing protease or an autocatalytic cleavage remains to be shown.