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J. Biol. Chem., Vol. 277, Issue 44, 41897-41905, November 1, 2002
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From the Department of Physiology, Tufts University School of Medicine, Boston, Massachusetts 02111
Received for publication, April 1, 2002, and in revised form, August 26, 2002
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Mannose phosphorylation of N-linked
oligosaccharides by UDP-GlcNAc:lysosomal enzyme
N-acetylglucosamine-1-phosphotransferase is a key step in
the targeting of lysosomal enzymes in mammalian cells and tissues. The
selectivity of this process is determined by lysine-based
phosphorylation signals shared by lysosomal enzymes of diverse
structure and function. By introducing new glycosylation sites at
several locations on the surface of mouse procathepsin L and modeling
oligosaccharide conformations for sites that are phosphorylated, it was
shown that the inherent flexibility of N-linked
oligosaccharides can account for the specificity of the transferase for
oligosaccharides at different locations on the protein. By using this
approach, the physical relationship between the lysine-based signal and
the site of phosphorylation of mannose residues was determined. The
analysis also revealed the existence of additional independent
lysine-based phosphorylation signals on procathepsin L, which account
for the low level of phosphorylation observed when the primary
Lys-54/Lys-99 signal is ablated. Mutagenesis of residues that surround
Lys-54 and Lys-99 and demonstration of mannose phosphorylation of a
glycosylated derivative of green fluorescent protein provide strong
evidence that the cathepsin L phosphorylation signal is a simple
structure composed of as few as two well placed lysine residues.
The mammalian lysosomal protein targeting system has
the capability of recognizing and modifying lysosomal hydrolases and growth factors from a wide range of protein families with high specificity. The molecular basis for this selectivity is due to the
activity of the UDP-GlcNAc:lysosomal enzyme
N-acetylglucosamine-1-phosphotransferase (GlcNAc-1-phosphotransferase), which phosphorylates N-linked
oligosaccharides of these proteins by the addition of GlcNAc-1-P
(1-5). This modification begins after lysosomal proteins are exported
from the endoplasmic reticulum and is followed by the removal of the
terminal GlcNAc moieties from the adducts. In the Golgi apparatus the
phosphorylated proteins are bound to mannose 6-phosphate receptors,
which mediate the delivery of the proteins to lysosomes.
GlcNAc-1-phosphotransferase has been purified as a 540-kDa complex
composed of disulfide-linked homodimers of Although the molecular basis for recognition of lysosomal hydrolases by
the transferase has been studied quite extensively, a complete
understanding of the nature of the recognition has remained elusive.
Early studies (9-12) demonstrated that the recognition involves
protein determinants that are conformation-dependent and
that the protein determinants in human cathepsin D extend over a large
portion of the surface of the protein and involve many residues
including lysine. We have shown that lysine residues alone can account
for most if not all of the energy of interaction between the
transferase and mouse cathepsin L and that lysine residues are the
major determinants for mannose phosphorylation of a wide range of
lysosomal proteins (13). Subsequently, we identified two lysine
residues, Lys-54 and Lys-99, as the ones involved in cathepsin L
phosphorylation (14). On the basis of these results, a relatively
simple model involving lysine residues was proposed as a general
phosphorylation signal for lysosomal proteins. Similar involvement of
lysine residues (15, 16) has since been demonstrated for other
proteins, and it is now widely accepted that lysine residues are the
primary determinants for mannose phosphorylation of a wide range of
proteins by the transferase.
Our recent studies have focused on determination of the molecular
dimensions of the phosphorylation signal and its relationship to the
oligosaccharides that are phosphorylated. With this information, it
will be possible to identify substrates for the transferase from
information in structural data bases and to engineer phosphorylation signals on macromolecules for therapeutic purposes. In our previous study (17), we compared lysine-based signals that were identified for
cathepsin L and cathepsin D, and we found that critical lysine residues
were separated by a similar distance (~34 Å) in the two proteins. In
this study, we further define the cathepsin L signal by determining its
relationship to the site of oligosaccharide phosphorylation. We also
examine the involvement of residues in the vicinity of critical lysine
residues and provide additional evidence for the simplicity and
generality of the signal.
Enzymes, Antibodies, cDNAs, and Other
Reagents--
Antibodies to mouse cathepsin L and green fluorescent
protein (GFP)1 were raised in
rabbits as described previously (18). LipofectAMINE PLUS was purchased
from Invitrogen. EcoScint H was acquired from National Diagnostics. The
QuikChange Mutagenesis Kit and Pfu Turbo were obtained from
Stratagene. DpnI and N-glycanase (peptide
N-glycosidase F) were purchased from New England Biolabs.
Other restriction enzymes were acquired from Invitrogen. Chemical
reagents were purchased from Sigma.
Cells and Growth Conditions--
COS-1 cells were purchased from
the American Type Culture Collection and were cultured in Dulbecco's
modified Eagle's medium (DMEM) supplemented with 10% fetal calf
serum, 100 units/ml penicillin, and 100 µg/µl streptomycin and
maintained at 37 °C in a humidified atmosphere of 5%
CO2.
Subcloning of Mouse Cathepsin L--
A fragment containing the
full coding sequence of mouse procathepsin L (19) was subcloned into
the pED4neo vector (20) for expression in COS-1 cells. The pED4neo
vector was modified by the insertion of a synthetic linker made from
the complementary oligonucleotides 5'-ATT TTC TCG AGA CCG GTG CGG CCG
CGA ATT CGT CGA CTC TAG AA-3' and 5'-AAT TTT CTA GAG TCG ACG AAT TCG
CGG CCG CAC CGG TCT CGA GA-3' into the EcoRI cloning site of
the vector. The linker contains XhoI, AgeI,
NotI, EcoRI, SalI, and XbaI
sites, in 5'-3' order, and eliminates the EcoRI site into
which it was cloned. The modified vector will hereby be referred to as
pED4neoSL. Mouse cathepsin L cDNA was ligated into the
XhoI and XbaI cloning sites to give an expression
vector, pED4neoSL-CL, suitable for transient or stable expression of
mouse cathepsin L in eukaryotic cells.
Mutagenesis of Cathepsin L cDNA--
All mutagenesis steps
were carried out using a modification of the QuikChange Site-directed
Mutagenesis kit (Stratagene). This method was chosen because of its
high mutation efficiency and because it requires no specialized
vectors. All oligonucleotide primers were designed using MutantMaker, a
Visual C++ application developed by one of the authors
(J. B. Warner), which expedites the task of primer selection. The
program reads in protein text files, generates mutagenic primers based
upon the user's parameters, and prints an oligonucleotide order form,
thereby minimizing human error. The following parameters were used in
designing the primers: GC content > = 40%, Tm > = 78 °C, and termination by G or C on both ends of the
oligonucleotide. Oligonucleotide primers were synthesized and purified
by SDS-PAGE. PCR was carried out using the following conditions: 1 cycle of 95 °C for 30 s, followed by 19 cycles of 95 °C for
30 s, 60 °C for 1 min, and 72 °C for 20 min. PCR products
were ethanol-precipitated, resuspended in deionized water, and
transformed into XL1-blue supercompetent cells (Stratagene). All
mutations were verified by sequencing.
Protein Expression and Quantitation of Phosphorylation--
COS
cells were transiently transfected with pED4neoSL-CL using the
LipofectAMINE PLUS reagent (Invitrogen) as described by the vendor. The
transfected cells were cultured for 48 h prior to labeling. The
cells were labeled for 6 h with 0.1 mCi/ml
[35S]methionine in DMEM containing 10% dialyzed fetal
calf serum, 0.9 mg/liter cold methionine, and 10 mM
NH4Cl, which causes the secretion of newly synthesized
lysosomal enzymes. Immunoprecipitation of cathepsin L from the media
and SDS-PAGE of labeled proteins were carried out as described
previously (21).
The methods used for quantitation of phosphorylation were similar to
those used previously (14, 17). Transfected cells were double-labeled
with 0.1 mCi/ml [3H]leucine and 0.4 mCi/ml
[32P]phosphate in DMEM containing 10% dialyzed fetal
calf serum and 10 mM NH4Cl. Unlabeled leucine
was reduced to 2% of the normal concentration to increase
incorporation of radioactive leucine into protein. Cathepsin L was
immunoprecipitated from the media and subjected to SDS-PAGE as
described previously except that unlabeled procathepsin L (4 µg) was
added as a carrier to each immunoprecipitate prior to SDS-PAGE. The
gels were stained with Coomassie Blue, and cathepsin L-containing bands
were excised from the gel and extracted by overnight incubation in 1 ml
of 1 N NaOH at room temperature. After neutralization with
1 ml of 1 N HCl, the amount of 32P and
3H in each band was determined by scintillation counting in
EcoScint H (National Diagnostics).
Phosphorylation of glyco-GFP was similarly quantitated, except that GFP
(4 µg) was added as a carrier to each immunoprecipitate prior to
SDS-PAGE. The difference in leucine usage between mouse procathepsin L
(19 residues) and glyco-GFP (20 residues) was taken into account when
comparing mannose phosphorylation of the two proteins.
Modeling of Procathepsin L Oligosaccharides--
A previously
modeled structure of mouse procathepsin L was used in these studies
(17). The Quanta molecular modeling program (Accelrys, Inc.) was used
to determine energetically acceptable conformations of a
Man8GlcNAc2 N-linked oligosaccharide
attached at various locations on the cathepsin L structure. The
Man8GlcNAc2 structure was used since in
vivo the terminal mannose in the middle branch of the
Man9GlcNAc2 oligosaccharide is clipped prior to phosphorylation of lysosomal proteins by GlcNAc-1-phosphotransferase (22). A set of acceptable conformations was generated for each oligosaccharide by randomly selecting torsion angles for all rotatable bonds in the Man8GlcNAc2 structure (Fig.
1), minimizing the potential energy of
the resulting structures and selecting those conformations that possess
an acceptable total potential energy value. A maximum potential energy
cut-off of 1000 kcal/mol was used that was found to be characteristic
of Man8GlcNAc2 conformations lacking clashes and close contacts. Fifty cycles of conjugate gradient minimization were used, which brought energy values of acceptable conformations close to convergence in most cases. Conformations were generated in the
context of the protein and torsion angles for rotatable bonds in the
oligosaccharide and in the asparagine side chain were randomly set.
Generally, less than 1% of the conformations that were generated met
the energy criteria. At least 2000 acceptable conformations were
obtained for each oligosaccharide. When the acceptable oligosaccharide
conformations were overlaid, they formed a spheroid-like object,
resembling a cloud. Therefore, such overlays of oligosaccharide
conformations are referred to as oligosaccharide clouds.
In order to assess the overlap between adjacent conformational clouds,
a specialized C++ application was developed. This application determines where selected residues in separate conformational clouds
can overlap in three-dimensional space. For each
Man8GlcNAc2 oligosaccharide there are five
mannose residues with a C-6-OH moiety, all of which are susceptible to
phosphorylation in vivo (22). Minima
(Xmin, Ymin, and
Zmin) and maxima (Xmax,
Ymax, and Zmax) are
determined for the coordinates of the O-6 atom of every
phosphorylatable mannose in each conformational cloud (Fig. 6). The
cube generated from these coordinates gives a simple but effective
definition of the boundaries of each conformational cloud. Boundaries
are generated for all clouds from phosphorylated mannoses. The program
then calculates the largest minimum and smallest maximum for each
coordinate to determine the overlapping region. If the free O-6 atom of
a mannose residue is contained in this overlapping region, its
coordinates are retained.
Generation of a Secreted, Glycosylated Form of GFP--
The
cDNA of proliferin-2, a secreted mannose 6-phosphate containing
glycoprotein, was purchased from American Type Culture Collection and
subcloned into the pED4neo expression vector. The signal sequence from
proliferin was amplified from pED4neo-proliferin using the following
PCR primers: 5'-CCG GAA TTC CGC ATG CTC CCT TCT TTG ATT CAA-3' (forward
primer) and 5'-TCC CCG CGG TGC ACA CAT GGG AAA TGA GGC-3' (reverse
primer). The PCR product was purified, cut with EcoRI and
SstII, and ligated into the EcoRI and
SstII cloning sites of the pEGFP-N1 vector
(Clontech). The modified GFP sequence was then
subcloned into the pED4neoSL vector using the XhoI and
NotI cloning sites. A glycosylation site was added to
residues 156-158 on the secretory GFP by site-directed mutagenesis, using the following primers: 5'-GTG AAG TTC GAG AAC GGC ACC CTG GTG AAC
CGC-3' (forward primer) and 5'-GCG GTT CAC CAG GGT GCC GTT CTC GAA CTT
CAC-3' (reverse primer). The location of the glycosylation site
positions the oligosaccharide on the rim the Topography of Mannose Phosphorylation of Cathepsin L--
To
examine the requirement for oligosaccharide placement, glycosylation
sites were placed at selected locations on the surface of cathepsin L
by site-directed mutagenesis, and glycosylation and mannose
phosphorylation at each site were determined after transient expression
in COS-1 cells. Thirty-three glycosylation site mutations spread
throughout the protein were created and tested. To maximize the number
of properly folded and glycosylated proteins, surface residues in
regions lacking
Wild-type procathepsin L contains two potential glycosylation sites,
only one (Asn-221) of which is utilized in the properly folded protein
(19). Because the cryptic site at Asn-268 can be glycosylated in some
modified cathepsin L proteins (19, 23), both the utilized site
(Asn-221) and the non-utilized site (Asn-268) were mutated to
glutamine, creating a construct encoding a nonglycosylated cathepsin L
protein. When expressed, the protein was secreted poorly as expected
(Fig. 2) since glycosylation is needed
for proper folding of cathepsin L in the COS-1 expression system (21). This construct was used for construction of the altered glycosylation site proteins listed in Table I.
Results on the synthesis, secretion, and glycosylation of selected
altered glycosylation site proteins are shown in Fig. 2, and data for
all of the constructs are summarized in Table I. Synthesis and
secretion of the proteins, as determined by biosynthetic labeling in
the presence of NH4Cl, was used as a means of assessing whether or not altered cathepsin proteins are folded properly. NH4Cl inhibits mannose 6-phosphate receptor function and
causes the quantitative secretion of newly synthesized
mannose-phosphorylated proteins. We have examined previously (13, 14,
17, 19, 21) more than 100 altered cathepsin L and cathepsin D proteins using the COS cell expression system. In all cases examined, normally folded proteins, as determined by level of catalytic activity (13),
susceptibility to proteolytic digestion (14, 17, 21), or susceptibility
to heat denaturation (13), are secreted under these conditions, whereas
abnormally folded proteins are retained within the cells and/or
degraded. Of the 33 altered glycosylation site proteins created for
this study, 19 were glycosylated and efficiently secreted (Fig. 2).
These were retained for further analysis. Endoglycosidase H (endo H)
treatment was used to examine the state of glycosylation of the
proteins (Table I). Oligosaccharides on proteins that undergo mannose
phosphorylation would be expected to remain sensitive to the treatment,
whereas those that do not would be expected to be further processed to
forms that are resistant to the treatment. Of the 19 retained proteins,
5 were sensitive to endo H treatment, 7 were partially sensitive, and 7 were resistant to the treatment.
Biosynthetic labeling with [32P]phosphate and
[3H]leucine was used to determine which of the altered
glycosylation site proteins were susceptible to mannose phosphorylation
(Fig. 3). Five of the constructs
(Asn-105, Asn-158, Asn-171, Asn-217, and Asn-229) displayed a high
level of phosphorylation approaching the phosphorylation level of
wild-type procathepsin L. All other constructs showed minimal (<25%)
phosphorylation compared with the wild-type protein. Phosphorylation of
the 5 highly phosphorylated constructs was inhibited by mutation of
Lys-54 and Lys-99 to alanine (Fig. 4) indicating that all 5 constructs utilize the previously identified mannose phosphorylation signal (13, 14).
The topographical locations of engineered glycosylation sites on the
surface of the protein are shown in Fig.
5. The locations of the glycosylation
sites of the highly phosphorylated constructs were found to be
clustered within the vicinity of Asn-221, the wild-type glycosylation
site. Most of the engineered sites in the vicinity of Asn-221 were
highly phosphorylated. Some sites in this region were phosphorylated
weakly (Asn-104, Asn-199, and Asn-208) or not at all (Asn-156 and
Asn-108). The low level of phosphorylation at these sites is attributed
to site-dependent effects that reduce accessibility of the
oligosaccharides to the transferase. Such effects would include
protein-oligosaccharide interactions that limit oligosaccharide
flexibility and site-dependent differences in
oligosaccharide processing that inhibit or prevent phosphorylation.
Flexibility of N-Linked Oligosaccharide Accounts for Topography of
Mannose Phosphorylation of Cathepsin L--
Previous studies (22, 24)
have shown that mannose-phosphorylated proteins display a surprising
heterogeneity with regard to sites of phosphorylation on the
oligosaccharide and the number and location of the phosphorylated
oligosaccharides on the protein. To account for this apparent lack of
specificity, we have proposed that selectivity of the reaction for
specific mannose 6-phosphate residues on protein-linked
oligosaccharides is dictated in large part by the ability of those
residues to migrate in three-dimensional space to the catalytic site of
GlcNAc-1-phosphotransferase when the protein is bound to the
transferase through its lysine-based phosphorylation signal (17). This
hypothesis is supported by NMR studies, which have indicated that
N-linked oligosaccharides are flexible in solution and that,
although preferred conformations may exist, such conformations are
short lived (25-28).
To test this hypothesis, three-dimensional oligosaccharide clouds
composed of compilations of randomly generated, energetically acceptable Man8GlcNAc2 oligosaccharide
conformations were modeled at phosphorylated glycosylation sites of
procathepsin L as described under "Experimental Procedures." The
oligosaccharide conformations that compose each cloud represent
sterically unhindered oligosaccharide conformations that were chosen
based on an energy value that would exclude clashes and close contact
of atoms within the oligosaccharide and between the oligosaccharide and
the protein. Thus, the volume enclosed by each cloud represents the
space available to one or more phosphorylatable mannose residues of the
oligosaccharide attached at that location on the protein. Clouds for
each glycosylation site are shown in Fig.
6. In these representations, only the
positions of O-6 atoms of phosphorylatable mannose residues are shown
(see Fig. 1). An overlay of all 6 clouds and a graphical representation of the overlap between clouds are also shown in Fig. 6. The existence of an overlap region shared by all 6 clouds substantiates the hypothesis described above by showing that all 6 oligosaccharides are
capable of positioning phosphorylatable mannose residues in the same
region in space. This region would correspond to the position of the
catalytic site of the transferase when it is bound to the protein.
Role of Lysine Residues in Residual Phosphorylation of Cathepsin
L--
The highly phosphorylated glycosylation sites including the
wild-type site (Fig. 4), as well as some of the weakly phosphorylated sites (data not shown), displayed significant levels of phosphorylation when lysine residues that compose the previously described
phosphorylation signal (Lys-54 and Lys-99) were mutated to alanine.
Whereas this phosphorylation represents a minor component of the
phosphorylation of the wild-type protein, it does represent a
substantial portion of the phosphorylation observed for two of the
engineered sites (Asn-105 and Asn-229). To determine whether or not
lysine residues are responsible for the residual phosphorylation of the
wild-type protein, site-directed mutagenesis was carried out on a
procathepsin L construct containing the wild-type glycosylation site
and alanine mutations at Lys-54 and Lys-99. The results shown in Fig.
7 indicate that several lysine residues,
including Lys-157, Lys-233, and Lys-237, contribute to this residual
phosphorylation. Phosphorylation of a construct containing alanine
mutations at Lys-54, Lys-99, Lys-116, Lys-157, Lys-233 and Lys-237 was
>90% inhibited, indicating that these lysine residues can account for
virtually all of the mannose phosphorylation of procathepsin L. The
residual phosphorylation appears to result from weak lysine-based
phosphorylation signals that act independently of the primary
Lys-54/Lys-99 signal. The level of residual phosphorylation differs
among the engineered glycosylation sites providing additional evidence
that this component of the phosphorylation results from independent
phosphorylation signals.
Role of Lysine Microenvironment in Mannose Phosphorylation of
Cathepsin L--
Surface residues within 8 Å of Lys-54 and Lys-99 on
wild-type cathepsin L were mutated to alanine in order to determine the importance of the environment surrounding these lysine residues (Table
II). No significant change in mannose
phosphorylation was observed in single alanine mutations; however, the
possibility remained that these residues had a weak interaction with
the transferase and worked in concert to provide a stronger contact. In
order to address this question, multiple mutations were created in
single constructs so that three residues surrounding Lys-54 (construct M56A/R57A/M58A) and five residues surrounding Lys-99 (construct H97A/Q98A/H100A/K101A/K102A) were mutated. In all cases there was no significant change in mannose phosphorylation of the cathepsin L
protein indicating that these residues play little if any role in the
mannose phosphorylation of cathepsin L.
Glycosylation and Mannose Phosphorylation of GFP--
To convert
GFP into a suitable substrate for the transferase, an endoplasmic
reticulum signal sequence and a glycosylation site were added as
described under "Experimental Procedures." This GFP construct,
called glyco-GFP, was completely glycosylated and secreted in COS-1
cells (Fig. 8A). When COS-1
cells expressing the construct were labeled with
[32P]phosphate, glyco-GFP was found to be phosphorylated
(Fig. 8B). Quantitation of mannose phosphorylation of
glyco-GFP was preformed using the [32P]phosphate and
[3H]leucine double-labeling protocol as described under
"Experimental Procedures." The level of phosphorylation was 16.8%
of that observed for wild type-cathepsin L but more than 8-fold greater
than background phosphorylation for this system (<2%) as determined
previously using glycopepsinogen (17). To determine whether the
phosphorylation was associated with the oligosaccharide, the labeled
protein was treated with specific endoglycosidases (Fig.
8B). N-Glycanase, which cleaves all
N-linked oligosaccharides, completely digested glyco-GFP and
removed all associated phosphorylation. Endo H, which reacts with only
high mannose oligosaccharides, cleaved only a small fraction of the
glycosylated protein, but removed phosphorylation completely,
indicating that the phosphorylation was located entirely on high
mannose oligosaccharides. Although actual residues that compose the
glyco-GFP phosphorylation signal were not elucidated, the high
concentration of surface lysine residues on this protein (20 in 239 amino acids) provides several pairs of suitably positioned lysine
residues that could serve as GlcNAc-1-phosphotransferase recognition
sites. The phosphorylation of a synthetic glycoprotein that could not
have evolved a complex phosphorylation signal provides evidence that
the signal is a relatively simple structure that may exist on a wide
range of proteins regardless of their need to be targeted to
lysosomes.
Finding a common structural motif for recognition of protein
substrates by GlcNAc-1-phosphotransferase has been a long-standing goal
in the field of lysosomal protein trafficking. Because the motif is
three-dimensional in nature and is found on proteins of diverse origin
and structure, it has been difficult to identify. Several studies have
been carried out to determine which amino acid residues are necessary
for mannose phosphorylation of individual proteins. Here, we provide
evidence that the inherent flexibility of N-linked
oligosaccharides enables these structures to be placed in a large
although limited area on a lysosomal protein and still have access to
the catalytic domain of the transferase. By placing oligosaccharides
throughout the surface of cathepsin L, it was possible to identify a
region in three-dimensional space where mannose residues are likely to
be phosphorylated and the spatial relationship of this site to lysine
residues Lys-54 and Lys-99 of the phosphorylation signal in the
protein. Knowing the placement of the oligosaccharide in reference to
the signal is necessary for identifying or constructing a signal that
not only allows interaction of the transferase with its substrate
protein but also provides access to its N-linked
oligosaccharides and phosphorylation of one or more mannose residues.
Findings of this study concerning the relationship of critical
components of the cathepsin L phosphorylation signal to the N-linked oligosaccharide of the protein are summarized in
Fig. 9. The distance of ~34 Å between
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
and 
subunits and two
identical, noncovalently associated 
subunits (6). The subunit
was shown to have nucleotide sugar binding activity, and on the basis
of previous genetic data, it was known that the catalytic and protein
recognition activities are likely to be located on separate subunits
(7). This has since been verified by analysis of the transferase in
cells from patients with mucolipidosis IIIC or variant pseudo-Hurler
polydystrophy. GlcNAc-1-phosphotransferase from these patients is
defective in the subunit, which prevents phosphorylation of
lysosomal enzymes, yet transferase activity on synthetic substrates is
retained (8).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Structure of the
Man8GlcNAc8 oligosaccharide. The
torsion angles that were randomized to generate oligosaccharide
structures are marked with a circular arrow. The locations
of the mannoses that can be phosphorylated are marked with an
asterisk (22). One terminal mannose residue was excluded
from this study since removal of this residue normally occurs prior to
mannose phosphorylation and is needed to allow phosphorylation of the
underlying mannose (22).
-barrel structure.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-helical structure were chosen for placement of the sites.
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Fig. 2.
Expression of cathepsin L glycosylation
mutants. The original glycosylation sites were removed from
wild-type procathepsin L, and new glycosylation sites were added by
site-directed mutagenesis. The indicated cathepsin L constructs were
expressed in COS-1 cells and split equally into two groups. One group
was labeled with [35S]methionine for 1 h. The other
group was similarly labeled for 1 h and chased for 6 h in
media containing unlabeled methionine. Cathepsin L was
immunoprecipitated from cell extracts of pulse-labeled
cells and from media of chased cells. Half of the
immunoprecipitated proteins were treated with endo H, and treated
and untreated immunoprecipitates were subjected to SDS-PAGE and
fluorography. A, fluorogram of untreated immunoprecipitates;
B, fluorogram of endo H-treated immunoprecipitates.
Glycosylated (G) and non-glycosylated (NG)
cathepsin L proteins are marked with arrows.
CL-ctrl represents the control procathepsin L, and
CL-ng represents the non-glycosylated procathepsin L
mutant.
Synthesis, secretion, and glycosylation of altered glycosylation site
constructs
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Fig. 3.
Mannose phosphorylation of procathepsin L
glycosylation mutants. The indicated cathepsin L constructs were
expressed in COS-1 cells and double-labeled with
[32P]phosphate and [3H]leucine for 6 h
in the presence of 10 mM NH4Cl. Procathepsin L
protein was immunoprecipitated from media and subjected to SDS-PAGE.
The protein bands were excised and quantitated in a scintillation
counter. The graph displays mannose phosphorylation as a percentage of
the wild-type cathepsin L (CL), and represents a typical
experiment with three replicates.
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Fig. 4.
Contribution of Lys-54/Lys-99 signal to
mannose phosphorylation of highly phosphorylated cathepsin L
constructs. Alanine mutations were introduced at Lys-54 and Lys-99
in each highly phosphorylated cathepsin L construct. Constructs with
and without these lysine mutations were expressed in COS-1 cells and
double-labeled with [32P]phosphate and
[3H]leucine for 6 h in the presence of 10 mM NH4Cl. Cathepsin L protein was
immunoprecipitated from the media and subjected to SDS-PAGE. The
labeled protein bands were excised and quantitated in a scintillation
counter. The graph displays mannose phosphorylation as a percentage of
the wild-type cathepsin L and represents the mean ± S.D. of two
experiments with a total of six replicates.
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Fig. 5.
Location of the new glycosylation
sites on cathepsin L. The wild-type glycosylation site (Asn-221)
is shown in blue. The glycosylation sites that were added
but were not significantly phosphorylated are shown in red.
Glycosylation sites that are highly phosphorylated are shown in
green. The -carbons of the critical lysines, Lys-54 and
Lys-99, are shown in yellow.
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Fig. 6.
Modeling of oligosaccharide clouds on
cathepsin L. Modeled structures for oligosaccharide clouds of
highly phosphorylated cathepsin L constructs are shown along with an
overlay and overlap images. The overlap image is a superposition of the
six individual structures. The overlap image shows the oligosaccharide
region shared by the six individual structures. Oligosaccharide clouds
were modeled as described under "Experimental Procedures." The
-carbons of critical lysine residues Lys-54 and Lys-99 are displayed
in white. WT, wild type.
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Fig. 7.
Alanine scanning mutagenesis of CL-K54A/K99A
construct. Lysine residues in the K54A/K99A construct were mutated
to alanine using site-directed mutagenesis. The cathepsin L constructs
were expressed in COS-1 cells and labeled with
[32P]phosphate and [3H]leucine for 6 h
in the presence of 10 mM NH4Cl. Cathepsin L
proteins were immunoprecipitated from media and subjected to SDS-PAGE.
The protein bands were excised and quantitated in a scintillation
counter. The graph displays mannose phosphorylation as a percentage of
the wild-type cathepsin L phosphorylation. The control represents the
K54A/K99A construct. The graph represents the mean ± S.D. of
three experiments with a total of nine replicates.
Effect of microenvironment surrounding critical lysines 54 and 99
View larger version (31K):
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Fig. 8.
Secretion and mannose
phosphorylation of glyco-GFP. Glyco-GFP and GFP were expressed in
COS-1 cells and labeled with [35S]methionine for 6 h
in the presence of 10 mM NH4Cl. The glyco-GFP
and GFP proteins were immunoprecipitated from cell extracts and media
and subjected to SDS-PAGE (A). Some of the
immunoprecipitates were incubated with endoglycosidase H (endo
H) or N-glycanase (B).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-carbon atoms of critical lysine residues appears to be a general
feature of the phosphorylation signal. This distance was demonstrated
in our earlier study (17) for cathepsin D as well as cathepsin L (14)
and is consistent with results obtained for two other proteins, DNase I
(15, 29) and aspartylglucosaminidase (16). In the case of
aspartylglucosaminidase, mutation of two lysine residues, Lys-183 in
the subunit and Lys-214 in the subunit, was found to inhibit
mannose phosphorylation by 96%. Because aspartylglucosaminidase is
expressed as a 22 heterotetramer with a
phosphorylated oligosaccharide in each subunit, these residues could be
used in one or more of a variety of configurations for phosphorylation.
Given this caveat, Lys-183 and Lys-214 are separated by 32.71 Å,
consistent with distances of 33.75 and 33.63 Å for cathepsin L and
cathepsin D, respectively (14, 17). Bovine DNase I has two
oligosaccharides and requires four lysine residues, Lys-27, Lys-50,
Lys-74, and Lys-124 for efficient mannose phosphorylation. Lys-27 and
Lys-74 have substantial effects on phosphorylation but do not appear to
belong to the same phosphorylation signal since they affect different
oligosaccharides. The two others residues, Lys-50 and Lys-124, are
located 34.31 Å apart, again consistent with the 34 Å inter-lysine
distance. The site of mannose phosphorylation for procathepsin L, which
was identified by determining the overlap of oligosaccharide clouds of
highly phosphorylated glycosylation sites, is also shown in Fig. 9. The
center of this region was calculated to be 26.83 Å from the closest
critical lysine, Lys-99. The relationship of the site of mannose
phosphorylation to the phosphorylation signal represents an intrinsic
property of GlcNAc-1-phosphotransferase and should apply to other
transferase substrates.
View larger version (38K):
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Fig. 9.
Model for mouse cathepsin L phosphorylation
signal. Murine procathepsin L is displayed along three different
axes to show the relationship between the critical lysine residues of
the protein and the mannose phosphorylation site (see Fig.
6H). The coordinate system is defined such that the Z plane
is the side of the protein that exposes the residues Lys-54 and Lys-99
(B), and the line between the residues delimits
the x axis. The distance between the Lys-99 and the midpoint
of the phosphorylation cloud was calculated as 26.83 Å. Note that
structures further from the viewer, such as the phosphorylation cloud
in the top panel, appear darker to indicate depth.
The results of this study suggest that the cathepsin L phosphorylation signal is polarized with one lysine residue (Lys-99) proximal to the site of mannose phosphorylation and the other lysine residue (Lys-54) distal. If the binding properties of these residues were equivalent, two regions of mannose phosphorylation corresponding to two transferase-binding orientations would be expected. Mutation of residues in the vicinity of critical lysine residues did not reveal involvement of other residues that would distinguish interaction of the two lysine residues with the transferase. However, it is possible that residues other than those tested could serve such a role. It is also possible that, because of the overall shape of procathepsin L, binding of the transferase to the cathepsin is sterically restricted to a single orientation or that binding in one orientation is nonproductive.
The existence of additional minor or cryptic lysine-based phosphorylation signals on cathepsin L is consistent with data gathered on other mannose-phosphorylated proteins. The chimeric studies of cathepsin D, as the authors note, can be interpreted by having two independent phosphorylation signals, instead of having a single extended signal (9). The existence of two or more signals on cathepsin D explains why mutation of individual lysine residues affects phosphorylation of the two cathepsin D oligosaccharides differently and why localized phosphorylation of engineered glycosylation sites was not observed for this protein (12, 15).
A previously unresolved issue concerning the nature of the phosphorylation signal is its level of complexity. Is the signal a complex, highly evolved structure that interacts with the transferase over an extended surface (29) or is it a relatively simple structure composed of a few well placed residues (17)? Two findings presented in this study address this issue. First, mutation of residues surrounding critical lysine residues to alanine had little if any effect on phosphorylation of cathepsin L. This supports our previous finding that lysine residues account for most if not all of the energy of interaction between the transferase and the cathepsin L phosphorylation signal (13, 14). These findings suggest that phosphorylation signals may actually be quite simple requiring as few as two well placed lysine residues as a minimal structure (14, 17). This does not mean that additional protein factors are not important in selected cases. Arginine has been shown to partially substitute for lysine in some contexts (15-17). Other residues in the vicinity of critical lysine residues may affect the accessibility or properties of the lysine residues. Effects such as this may explain the apparent involvement of tyrosine residues in phosphorylation of aspartylglucosaminidase and DNase I (16, 29). In theory, any residue in contact with the transferase when it is bound to the protein could affect the rate and efficiency of phosphorylation. The issue of complexity is also addressed by the finding that glyco-GFP is susceptible to mannose phosphorylation. Given the origin, subcellular location, and structure of GFP, there is no logical explanation for how a complex mannose phosphorylation signal could have evolved on this protein. On the other hand, phosphorylation of this protein is completely compatible with a simple phosphorylation signal composed of a few well placed lysine residues.
Finally, a simple phosphorylation signal is compatible with what is
known about the evolution of the mannose 6-phosphate recognition system. Utilization of the mannose 6-phosphate recognition system for
lysosomal targeting was a relatively late event in evolution probably
occurring sometime during early vertebrate evolution. Evolution of the
system would have required a genetic mechanism for generating mannose
phosphorylation signals on a structurally diverse set of lysosomal
hydrolases. Generation of a complex structure on a diverse set of
proteins would have required extensive remodeling of the proteins. If
such alterations had occurred, one would expect to observe
manifestations of these changes when comparing hydrolase sequences from
species that utilize the mannose 6-phosphate recognition system and
those that do not. Such manifestations have not been observed. A simple
phosphorylation signal, such as the one described, would allow
generation of phosphorylation signals on proteins of diverse structure
through one or two point mutations and would be fully compatible with
evolution of the system.
| |
ACKNOWLEDGEMENTS |
|---|
We thank Laura Liscum and Michael Forgac for critical review of the manuscript.
| |
FOOTNOTES |
|---|
* This work was supported by National Institutes of Health Grant CA66575.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The atomic coordinates and the structure factors (code 1MVV) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).
To whom correspondence should be addressed: Dept. of Physiology,
Tufts University School of Medicine, 136 Harrison Ave., Boston, MA
02111. Tel.: 617-636-6748; Fax: 617-636-0445; E-mail: gary.sahagian@tufts.edu.
Published, JBC Papers in Press, August 28, 2002, DOI 10.1074/jbc.M203097200
| |
ABBREVIATIONS |
|---|
The abbreviations used are: GFP, green fluorescent protein; DMEM, Dulbecco's modified Eagle's medium; endo H, endoglycosidase H.
| |
REFERENCES |
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RESULTS
DISCUSSION
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