Research
© 2013 by The American Society for Biochemistry and Molecular Biology, Inc.
This paper is available on line at http://www.mcponline.org
Extending the Mannose 6-Phosphate
Glycoproteome by High Resolution/Accuracy
Mass Spectrometry Analysis of Control and
Acid Phosphatase 5-Deficient Mice*□
S
David E. Sleat‡§¶, Pengling Sun‡, Jennifer A. Wiseman‡, Ling Huang‡§,
Mukarram El-Banna‡, Haiyan Zheng‡, Dirk F. Moore㥋, and Peter Lobel‡§¶
In mammals, most newly synthesized lumenal lysosomal
proteins are delivered to the lysosome by the mannose
6-phosphate (Man6P) targeting pathway. Man6P -containing proteins can be affinity-purified and characterized using proteomic approaches, and such studies have led to
the discovery of new lysosomal proteins and associated
human disease genes. One limitation to this approach is
that in most cell types the Man6P modification is rapidly
removed by acid phosphatase 5 (ACP5) after proteins are
targeted to the lysosome, and thus, some lysosomal proteins may escape detection. In this study, we have extended the analysis of the lysosomal proteome using high
resolution/accuracy mass spectrometry to identify and
quantify proteins in a combined analysis of control and
ACP5-deficient mice. To identify Man6P glycoproteins
with limited tissue distribution, we analyzed multiple tissues and used statistical approaches to identify proteins
that are purified with high specificity. In addition to 68
known Man6P glycoproteins, 165 other murine proteins
were identified that may contain Man6P and may thus
represent novel lysosomal residents. For four of these
lysosomal candidates, (lactoperoxidase, phospholipase D
family member 3, ribonuclease 6, and serum amyloid P
component), we demonstrate lysosomal residence based
on the colocalization of fluorescent fusion proteins with a
lysosomal marker. Molecular & Cellular Proteomics 12:
10.1074/mcp.M112.026179, 1806 –1817, 2013.
The lysosome is a key site within the cell for the digestion of
macromolecules, including proteins, carbohydrates, lipids,
and nucleic acids (1), and this catabolic function is enabled by
the concerted action of numerous hydrolases that have
From the ‡Center for Advanced Biotechnology and Medicine, Piscataway, the §Department of Biochemistry and Molecular Biology,
Robert-Wood Johnson Medical School, University of Medicine and
Dentistry of New Jersey, and the 㛳Department of Biostatistics, University of Medicine and Dentistry of New Jersey-School of Public
Health, Piscataway, New Jersey 08854
Received November 27, 2012, and in revised form, March 5, 2013
Published, MCP Papers in Press, March 11, 2013, DOI
10.1074/mcp.M112.026179
1806
evolved to function in the acidic environment of the lysosome.
Most lysosomal hydrolases and their accessory proteins are
soluble and located within the lumen of the lysosome, and to
date, ⬃70 such proteins have been identified (2, 3). Mutations
in the genes encoding more than 30 of these proteins result in
lysosomal storage diseases in which unhydrolyzed substrates
accumulate within lysosomes, disrupting cellular function and
frequently resulting in cell death (32).
Most newly synthesized soluble, lumenal lysosomal proteins are directed to the lysosome via the mannose 6-phosphate (Man6P)1 pathway (4). Here, select N-linked carbohydrates receive the Man6P modification that is recognized by
two Man6P receptors (MPRs) that direct the vesicular trafficking of the lysosomal proteins from the trans-Golgi to an acidified prelysosomal compartment. In most cell types, the
Man6P modification is rapidly removed by acid phosphatase
5 (ACP5) (5) upon arrival within the lysosome.
The presence of Man6P provides a unique opportunity for
the study of lysosomal proteins by allowing for the highly
specific affinity purification of this class of proteins from complex mixtures using MPRs immobilized on a solid affinity
support (6). Man6P-containing lysosomal proteins have been
purified from a wide variety of biological sources, and their
characterization by proteomic methods has provided valuable
insights into the composition and function of the lysosome as
well as the roles of lysosomal proteins in human disease (2, 7).
In mammals, the tissue distribution of lysosomal proteins can
be highly restricted, and thus one approach that has effectively increased proteome coverage has been to analyze multiple individual tissues (3).
1
The abbreviations used are: Man6P, mannose 6-phosphate;
ACP5, acid phosphatase 5; LPO, lactoperoxidase; PLD3, phospholipase D family member 3; RNASE6, ribonuclease 6; APCS, serum
amyloid P component; MPR, mannose 6-phosphate receptor; sCIMPR, soluble cation-independent mannose 6-phosphate receptor;
PBS-I, PBS with inhibitors; PBS-TI, PBS with inhibitors and Tween 20;
Glc6P/Man, glucose 6-phosphate and mannose; GLB1L, ␤-galactosidase 1-like; ER, endoplasmic reticulum; SLRRP, small leucine-rich
repeat proteoglycan.
Molecular & Cellular Proteomics 12.7
Mannose 6-Phosphate Glycoproteome
One limitation to the MPR affinity purification approach is
that in most cells and tissues, a relatively small fraction of
lysosomal proteins contain Man6P (8) because of natural dephosphorylation by ACP5 (5), with the notable exception of
brain (9). As a result, minor lysosomal components or those
that are particularly susceptible to dephosphorylation, either
in vivo or during the course of purification, may be overlooked
in proteomic surveys. In addition, as the proportion of Man6P
glycoproteins in the complex mixture of proteins of an initial
tissue extract becomes smaller, the proportion of contaminants is likely to increase in the purified preparation. In practical terms, extensive dephosphorylation of lysosomal proteins can make purification from limiting tissue sources in
whole organism surveys a difficult or impossible prospect.
In this study, we have extended the proteomic analysis of
mammalian Man6P glycoproteins with an analysis of multiple
tissues from the mouse. We address the problem of dephosphorylation of lysosomal proteins with the inclusion of multiple
tissues from ACP5-deficient mice (10), and we apply high
resolution/accuracy mass spectrometry to confidently identify
components of the mixture. Sixty eight known Man6P glycoproteins (65 lysosomal and 3 nonlysosomal) are identified,
which is comparable with earlier studies. However, the modified approach has also resulted in the identification of 165
other murine proteins that are specifically purified by MPR
affinity chromatography and that may potentially represent
lysosomal proteins. This is the most extensive list obtained to
date for candidate mannose 6-phosphorylated proteins, and
we demonstrate that four of these proteins localize to the
lysosome using morphological methods.
EXPERIMENTAL PROCEDURES
Animals—Experiments and procedures involving live animals were
conducted in compliance with protocols approved by the Robert
Wood Johnson Medical School Institutional Animal Care and Use
Committee. Acp5⫺/⫺ mutant mice have been described previously
(10) and were in an isogenic 129SvEv genetic background. Mice were
deeply anesthetized with a mixture of sodium pentobarbital and sodium phenytoin (Euthasol, Delmarva Laboratories, Milton, DE) and
killed by exsanguination following transcardiac perfusion with saline.
Tissues were removed by dissection, frozen on dry ice, and stored at
⫺80°C prior to use.
Purification of Man6P Glycoproteins—Proteins containing Man6P
were isolated by affinity purification on immobilized bovine soluble
cation-independent MPR (sCI-MPR) columns using a modification of
a procedure originally developed for rat tissues (3). For Experiments
1–3 (supplemental Table 1), organs from multiple animals were dissected and pooled, and a maximum of 5 g of a given organ type were
used for the purification of Man6P glycoproteins. Small organs for
which the aggregate pool weighed 5 g or less were homogenized
directly. Large organs (e.g. liver) were frozen, powdered with a Bessman tissue pulverizer (Spectrum Laboratories, Rancho Dominguez,
CA), pooled together, and mixed, and 5 g were removed for homogenization. Tissues were homogenized on ice using a Polytron (Brinkmann Instruments) in 100 ml of PBS-TI (PBS containing 0.2% Tween
20, 2.5 mM EDTA, 5 mM ␤-glycerophosphate, 1 ␮g/ml pepstatin, 1
␮g/ml leupeptin, and 0.5 mM Pefabloc). Carcasses were homogenized and analyzed individually. Each carcass (⬃20 g) was individually
Molecular & Cellular Proteomics 12.7
homogenized in 200 ml of buffer, and 50 ml of homogenate was
removed and added to 50 ml of fresh buffer to maintain a volume/
weight ratio of ⬎20 as used for other tissues. Homogenates were
centrifuged at 15,000 ⫻ g for 45 min at 4°C, filtered through Whatman
No. 1 paper, and then loaded onto a 5-ml bed volume of immobilized
sCI-MPR at a rate of 50 ml/h. Columns were washed with 30 ml of
PBS-TI at 10 ml/h, gravity flow batch-washed four times with 10 ml of
PBS-TI, then four times with 10 ml of PBS-I (i.e. PBS-TI without
Tween 20), and then washed overnight with 80 ml PBS-I at 5 ml/hr.
Columns were gravity flow-eluted two times with 5 ml of PBS containing 5 mM glucose 6-phosphate and 5 mM mannose (Glc6P/Man)
and then two times with 5 ml of PBS containing 5 mM Man6P. Eluates
were concentrated using Amicon Ultra-4 Ultracel-10k filtration devices. For Experiment 4, Man6P glycoproteins were affinity-purified
from individual wild-type mouse brains as described previously (12).
Mass Spectrometry—Samples of Man6P eluates and equivalent
proportions of the total eluate of the corresponding Glc6P/Man eluates were heated for 10 min at 60°C in reducing, denaturing SDSPAGE sample buffer and then fractionated on precast 10% polyacrylamide gels (Invitrogen) until the bromphenol blue dye front had run ⬃1
cm into the gel (3). Gel slices corresponding to each sample were
excised and cut into small pieces, reduced, alkylated with iodoacetamide, and digested with trypsin (specificity, carboxyl side of lysine
and arginine) using standard methods (13). Samples were analyzed in
duplicate using an LTQ Orbitrap Velos tandem mass spectrometer
coupled to a Dionex Ultimate 3000 RLSCnano System (Thermo Scientific, Somerset, NJ). Digests were solubilized in 0.1% TFA, and 0.5
␮g were loaded on to a fused silica trap column of 100 ␮m ⫻ 2 cm
packed with Magic C18 AQ (5 ␮m bead size, 200-Å pore size; BrukerMichrom, Auburn, CA). The trap column was washed with 0.2%
formic acid at a flow rate of 10 ␮l/min for 5 min. Retained peptides
were separated on a fused silica column of 75 ␮m ⫻ 50 cm packed
with Magic C18 AQ (3-␮m bead size, 200-Å pore size; BrukerMichrom) using a segmented linear gradient from 4 to 90% B (A: 0.1%
formic acid, B: 0.08% formic acid, 80% ACN): 5 min, 4 –10% B; 60
min, 10 – 40% B; 15 min, 40 –55% B; 10 min, 55–90% B. For each
cycle, one full MS was scanned in the Orbitrap with resolution of
60,000 from 300 to 2000 m/z, and the 20 most intense peaks were
fragmented by CID using a normalized collision energy of 35%, and
products were scanned in the ion trap. Data-dependent acquisition
was set for a repeat count of 2 and exclusion of 60 s.
Generation of Peak Lists—Peak lists were generated using Proteome Discoverer 1.2 with minimum and maximum precursor masses
of 350 and 6000 Da, respectively, minimum signal/noise of 1.5, and
no constraints with respect to retention time, charge state, or peak
count.
Database Search—A local implementation of the Global Proteome
Machine (14, 15) Cyclone XE (Beavis Informatics Ltd., Winnipeg,
Canada) with X!Tandem version CYCLONE (2011.12.01) was used to
search mass spectrometry data. Data from murine samples analyzed
using the Orbitrap Velos were searched against ENSEMBL release 64
of the NCBIM37 mouse genome assembly (21886 known genes)
together with a contaminant database containing common contaminant proteins and the bovine cation-independent mannose-6-phosphate receptor precursor (gi兩27805933). The following search parameters were used: fragment mass error, 0.4 Da; parent mass error, 10
ppm; maximum charge, ⫹4; minimum 15 peaks assigned; 1 missed
cleavage allowed. Carbamidomethylation was a constant modification; methionine oxidation was a variable modification throughout the
search; and tryptophan oxidation, asparagine deamidation, and glutamine deamidation were variable modifications during model refinement. MS data files were analyzed together in a MudPit analysis and
individual data extracted. ENSEMBL protein identifiers were converted to associated gene names to collapse multiple protein identi-
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Mannose 6-Phosphate Glycoproteome
fiers that can be assigned to the same gene. Assignments were
filtered for a log Global Proteome Machine expectation score of ⫺10
or better and a minimum of two unique peptides were identified. Note
there are a small number of proteins that are labeled as “acceptable
gene product assignments” with a single unique peptide. These represent cases in which a protein identifier with a single unique peptide
maps, together with other protein identifiers, to a gene identifier that
is represented by two or more unique peptides in total. LTQ data from
rat samples (3) were searched as described above except that the
parent ion mass error was ⫹4 to ⫺0.5 Da and that the ENSEMBL,
Refseq, Uniprot, and IPI rat protein databases were used instead of
the mouse database. Database locations and creation dates were as
follows: ENSEMBL, ftp.ensembl.org/pub/release-68/fasta/rattus_
norvegicus/pep/Rattus_norvegicus.RGSC3.4.68.pep.all.fa.gz, 7/20/
2012; Refseq, ftp.ncbi.nlm.nih.gov/genomes/R_norvegicus/protein/
protein.fa.gz 6/20/12; Uniprot; ftp.uniprot.org/pub/databases/
uniprot/current_release/knowledgebase/proteomes/Rat.fasta.gz
7/11/12; and IPI ftp://ftp.ebi.ac.uk/pub/databases/IPI/current/
ipi.RAT.fasta.gz, 7/20/12.
Localization of Candidate Lysosomal Proteins—The subcellular localization of candidate lysosomal proteins was determined using
C-terminally tagged fusions with mCherry, a marker that is fluorescent
at lysosomal pH and refractory to degradation (16). Constructs were
based on pmCherry-N1 (Clontech) and were designed to remove the
11 N-terminal residues of mCherry and all linker sequences between
the candidate and the fluorescent tag. CHO cells were transfected
using Lipofectamine 2000 (Invitrogen) and a mixed population of
stably transfected cells obtained by selection with puromycin (Invitrogen). Cells were plated on glass coverslips and treated for 24 h with
AlexaFluor-488 (Invitrogen)-labeled recombinant human NPC2, a lysosomal protein that can be delivered to the lysosome by MPRmediated endocytosis (17). Images were acquired using a Zeiss LSM
700 (Jena, Germany) confocal scanning laser microscope with a ⫻63
water immersion lens. Quantitative analysis of colocalization of candidate-mCherry fusion proteins with the lysosomal marker was conducted using the Colocalization Threshold plugin of ImageJ (18).
RESULTS AND DISCUSSION
Experimental Design—The loss of ACP5 results in increased steady-state levels of Man6P glycoproteins in many
tissues; thus, the analysis of Acp5⫺/⫺ mice could potentially
increase the sensitivity of Man6P glycoproteomics studies in
two ways. First, we may detect proteins that are of low abundance and/or readily dephosphorylated. Second, the greater
yield of Man6P glycoproteins would decrease the relative
levels of contaminants detected during MS/MS analysis. This
is important given that a major challenge is differentiating
contaminants from proteins of interest.
Previously, we recognized that there are two classes of
contaminant in Man6P glycoprotein purification schemes (3,
13). Specific contaminants elute from the affinity media in the
presence of Man6P; these represent proteins that do not
contain Man6P but interact with true Man6P glycoproteins.
Examples include cystatins, some of which are found in
preparations of purified Man6P glycoproteins (13) despite
their lack of glycosylation and whose presence can be
explained by copurification with lysosomal cysteine proteases. Nonspecific contaminants are proteins that are retained within the affinity column after washing via Man6Pindependent interactions.
1808
Our experiments are designed to distinguish between nonspecific contaminants and true Man6P glycoproteins and associated proteins. For each sample, after washing, we first
elute the affinity column with a mixture of glucose 6-phosphate and mannose (Glc6P/Man). This mock elution should
contain some nonspecifically bound proteins that are slowly
leaching off the column as well as displacing some lectins. We
then elute the column with Man6P. This specific elution releases proteins bound to the immobilized sCI-MPR via
Man6P-dependent interactions. The relative amounts of
Man6P glycoproteins and nonspecific contaminants should
be increased in the Man6P eluate and decreased in the
Glc6P/Man eluate. Thus, a given protein may be classified
following mass spectrometric analysis by comparing its spectral counts in the two eluates, and we have developed statistical methods for this purpose to provide a point estimate and
confidence interval for degree of enrichment (3) together with
a probability (i.e. a q value that is the p value corrected for
multiple measurements). In this way, we classify proteins as
clear contaminants (higher in the Glc6P/Man than Man6P
eluate, q ⱕ0.05), potential Man6P glycoproteins (higher in the
Man6P than Glc6P/Man eluate, q ⱕ0.05), or ambiguous (e.g.
q ⬎0.05). In the mass spectrometric analysis of the two eluates, we use the same proportion of the total Glc6P/Man and
Man6P eluates rather than equal amounts of protein. It is
worth noting that the amount of sample analyzed for the
Man6P eluate likely lies outside the linear range in terms of
spectral count response (data not shown). As a result, we may
be underestimating total spectral counts obtained in the
Man6P eluate and thus underestimating the degree of enrichment in this eluate compared with the Glc6P/Man eluate. In
effect, this means that our results may be subject to conservative error (i.e. some proteins that are enriched in the Man6P
eluate may be classified as ambiguous).
The study was conducted as a series of four independent
experiments. These are summarized in supplemental Table 1
together with identifiers for the 116 individual mass spectrometry runs and database searches. Experiment 1 was a direct
comparison of proteins purified from wild-type and Acp5⫺/⫺
liver. Experiment 2 was an initial analysis of total Acp5⫺/⫺
mouse homogenate as well as six individual tissues (brain,
heart, kidney, liver, small intestine, and spleen). Experiment 3
was an expanded analysis of Acp5⫺/⫺ tissues (adrenal, brain,
epididymis, heart, kidney, large intestine, liver, lung, ovary,
small intestine, spleen, stomach, testes, and thymus) as well
as the remaining carcass. Finally, Experiment 4 was an analysis of wild-type brain. We also conducted a reanalysis of a
previous study of Man6P glycoproteins from multiple rat tissues (3) using more current rat protein databases as an additional validation of candidates arising from the analysis of
mouse samples.
Analysis of Man6P Glycoproteins Purified from Control and
Acp5⫺/⫺ Liver—We examined the distribution of proteins in
preparations of Man6P glycoproteins from liver from both
Molecular & Cellular Proteomics 12.7
Mannose 6-Phosphate Glycoproteome
FIG. 1. Assignment of spectral counts in affinity column eluates
from Acp5ⴙ/ⴙ and Acp5ⴚ/ⴚ mouse liver. Samples were purified
from pooled total liver from 3 to 5 animals (Experiment 1). Spectral
counts of known Man6P (filled bars) or other mouse proteins (open
bars) are the mean of duplicate LC-MS/MS analyses with error bars
representing the range.
wild-type and ACP5-deficient mice (supplemental Table 2).
Proteins were classified as either “known Man6P” (2) or
“other,” a category representing murine proteins that are not
currently assigned to this organelle. Note our database of
known Man6P category (supplemental Table 3) mainly includes characterized lysosomal proteins but also includes
several proteins (myeloperoxidase, deoxyribonuclease 1, and
renin) that are not considered primarily lysosomal. We classify
granzymes A and B within the lysosomal category as they
reside in specialized lysosome-related organelles. The other
category includes contaminants as well as the novel lysosomal proteins whose identification represents the primary
aim of this study. ACP5 is expressed at high levels in liver, and
its loss results in greatly increased levels of Man6P glycoproteins within this organ (5). Although spectral counts assigned
to known Man6P glycoproteins were similar in wild-type and
mutant liver for both Man6P and Glc6P/Man eluates, the
relative level of other proteins was greatly decreased in the
absence of ACP5 (Fig. 1, Table 1). This demonstrates that, as
predicted, reducing natural dephosphorylation increases the
relative selectivity of purification of Man6P glycoproteins.
FIG. 2. Spectral counts assigned to individual known Man6P
glycoproteins in Man6P eluates of Acp5ⴚ/ⴚ and Acp5ⴙ/ⴙ mouse
liver. Data represent sum of spectral counts of duplicate LC-MS/MS
runs of each sample analyzed in Experiment 1. Only proteins with ⱖ20
spectral counts when combining Acp5⫺/⫺ and wild-type samples are
shown. Note that ACP5 is indicated with the filled circle.
It is important to consider the effect of ACP5 on expression
of the Man6P-containing forms of lysosomal proteins. In Fig.
2, we compared spectral counts assigned to known Man6P
glycoproteins in the Man6P eluates from wild-type and ACP5deficient mouse liver. We find that the levels of most known
Man6P glycoproteins were elevated in the Acp5⫺/⫺ mouse
liver. However, in addition to ACP5 itself, several proteins
were markedly decreased, including ␤-glucuronidase, prosaposin, cathepsins B, O, and S, and myeloperoxidase (supplemental Table 2). In view of this, our overall strategy to identify
novel lysosomal candidates was modified to include liver and
brain from wild-type animals in addition to the multiple organs
from the Acp5⫺/⫺ mice.
Analysis of Multiple Acp5⫺/⫺ Mouse Tissues—Fig. 3 shows
the relative levels based on spectral counting of lysosomal
and other proteins present in the Glc6P/Man and Man6P
eluates from the affinity chromatography of extracts of multiple organs from Acp5⫺/⫺ mice as well as wild-type brain.
Total spectral counts were lower in the Glc6P/Man eluates
TABLE I
Ratio of spectral counts in Man6P compared with Glc6P/Man eluates in purified samples from Acp5⫹/⫹ and Acp5⫺/⫺ mouse liver
Protein assignments are classified as significantly elevated (q ⱕ0.05), significantly decreased (q ⱕ0.05), or unclassified (q ⬎0.05), where low
q values (which are derived from p values) indicate low false discovery rates (11). Data are derived from duplicate LC-MS/MS analyses of 3–5
livers pooled for each genotype and filtered for proteins with total spectral counts (Man6P eluate plus Glc6P/Man eluate) of ⱖ20 per protein.
Genotype
Protein class
Specificity of purification
Depleted
Total
Acp5⫺/⫺
Known Man6P
other
52
39
2
25
0
17
54
88
Acp5⫹/⫹
Known Man6P
other
49
41
7
68
1
106
57
222
Molecular & Cellular Proteomics 12.7
Enriched
Ambiguous
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Mannose 6-Phosphate Glycoproteome
FIG. 3. Spectral counts assigned in affinity chromatography eluates. Data show the mean of spectral count data from two replicate
LC-MS runs of each purified sample with error bars representing the range. Contaminants of non-murine origin are excluded. All tissues are
from Acp5⫺/⫺ animals except for the indicated wild-type brains. Panel A, total spectral counts assigned in Man6P and Glc6P/Man eluates;
panel B, spectral counts assigned to lysosomal and other proteins in Man6P eluates; panel C, spectral counts assigned to lysosomal and other
proteins in Glc6P/Man eluates. Note that in some cases, in the Glc6P/Man eluate, levels of proteins not known to contain Man6P glycoproteins
were significantly higher than other tissues (e.g. panel C, Brain Expt. 2 and Liver Expt. 3), possibly indicating that initial washing conditions were
not sufficiently stringent to remove contaminants. However, in the Man6P eluate (panel B), these proteins were similar to those in other organs,
indicating that the Glc6P/Man elution provided a final and effective wash.
1810
Molecular & Cellular Proteomics 12.7
Mannose 6-Phosphate Glycoproteome
compared with the Man6P eluate in most organs (Fig. 3,
panel A). Analysis of the Man6P eluates revealed that most
of the spectral counts were assigned to known Man6P
glycoproteins (Fig. 3, panel B), the vast majority being lysosomal. In contrast, analysis of the Glc6P/Man eluates
revealed that the majority of spectral counts were assigned
to proteins not established to contain Man6P (Fig. 3, panel
C). Levels of such proteins overall were relatively unchanged in the two eluates or depleted in the Man6P eluate,
with the mean amount in the Man6P eluate being 0.8-fold
that of the Glc6P/Man eluate (Fig. 3, compare panels B and
C). For known Man6P glycoproteins, levels were on average
⬃5-fold higher in the Man6P eluate than in the Glc6P/Man
eluate.
Identification of Candidate Man6P Glycoproteins—Our approach to the identification of novel lysosomal and other
Man6P glycoproteins from mouse was to analyze data obtained for all experiments (supplemental Table 1), including
multiple tissues from ACP5-deficient animals and liver and
brain from wild-type controls. We used a MudPIT style,
merged database search strategy and identified candidates
by measuring the specificity of purification as described
above (i.e. by comparing spectral counts obtained for each
protein in the Glc6P/Man and Man6P eluates). In total, we
identified 68 Man6P-containing proteins that included 65
known lysosomal proteins. In addition, we identified 1292
mouse proteins that are not currently thought to contain
Man6P (protein and peptide assignments are shown in supplemental Tables 4 and 5, respectively). In Fig. 4, panel A, we
compare the distribution of spectral counts assigned to each
protein in the Glc6P/Man and Man6P eluates. Note that for
the purposes of this analysis, data were filtered to accept only
murine proteins with total spectral counts of ⱖ20, yielding 68
and 633 known Man6P and other proteins, respectively.
(Identity and statistical analysis for these are listed in supplemental Table 6.)
We have evaluated the experiment to experiment variation of the relative levels of individual proteins in Man6P
compared with the Glc6P/Man eluates, and these analyses
are included in supplemental material. The supplemental
Figs. 1– 4 are heatmap diagrams that provide a global visual
evaluation of experimental variation for each individual protein. The supplemental Figs. 5– 8 are forest plots (19) depicting the proportion of individual proteins in Man6P compared with the total (Man6P plus Glc6P/Man) eluates, with
bars indicating 95% percentile confidence, and plot box
areas being proportional to this confidence. For both
graphic depictions, protein assignments are categorized as
known Man6P (supplemental Figs. 1 and 5), and proteins
that are not known to contain Man6P but are either enriched
in the Man6P eluate (supplemental Figs. 2 and 6), cannot be
classified in terms of specificity of purification (supplemental Figs. 3 and 7), or are depleted in the Man6P eluate
(supplemental Figs. 4 and 8).
Molecular & Cellular Proteomics 12.7
All known Man6P glycoproteins were significantly enriched
in the Man6P compared with the Glc6P/Man eluate with the
exception of GM2 activator protein (GM2A), which was depleted. This may indicate that GM2A is a particularly low
affinity ligand for the sCI-MPR or that it is purified in physical
association with another Man6P glycoprotein, a possibility
supported by the fact that its intracellular targeting is only
partly dependent upon Man6P (20).
Six hundred and thirty three proteins that are not currently
thought to contain Man6P were identified that met the threshold for total spectral counts. Of these, most (363) were significantly depleted in the Man6P eluate and are thus likely to
represent contaminants. For 105 proteins, no significant conclusions could be drawn regarding distribution in the two
eluates. However, 165 proteins were significantly elevated in
the Man6P eluate, potentially indicating the presence of
Man6P.
As an additional level of validation, we compared results
obtained with mouse with a reanalysis of earlier data obtained
from a study of multiple rat tissues (3) (protein and peptide
assignments and statistical analyses are shown in supplemental Tables 7–9). The number (65) and degree of enrichment of known Man6P proteins was similar to that observed
from mouse, with GM2A also being depleted in the specific
eluate (Fig. 4, panel B). Two hundred and twenty five other rat
proteins not known to contain Man6P that met the threshold
for spectral counts (ⱖ20) were detected, a number considerably less than the 633 found with mouse. This may reflect the
number of LC-MS/MS analyses (116 for mouse and 67 for rat)
as well as differences in instrumentation (Orbitrap Velos for
mouse versus LTQ for rat). Nonetheless, we can use the
overlapping datasets to further test the validity of assignments. In total, 209 proteins were identified in both species,
including 64 known Man6P glycoproteins. In general, classification of proteins that were identified in both rat and mouse
corresponded well, with 174/209 proteins classified similarly
for the two species (Table II). This agreement is also reflected
by the correlation between enrichment factors for proteins
identified in preparations from mouse and rat (Fig. 4, panel C).
Identification and Validation of Lysosomal Candidates—
Candidate Man6P glycoproteins identified from the aggregate
analysis of all samples are broadly classified in Table III and
listed in detail in supplemental Table 10. We selected four
lysosomal candidates (serum amyloid P component (APCS),
lactoperoxidase (LPO), phospholipase D family, member 3
(PLD3), and ribonuclease k6 (RNASE6)) to evaluate subcellular distribution. Our approach was to express fluorescent
fusions between mCherry and the candidates of interest and
compare cellular distribution with either a fluorescent-labeled
NPC2 protein or a fluorescent-labeled dextran that are both
endocytosed and delivered to the lysosome. These analyses
were conducted in either stably transfected CHO cells (Fig. 5)
or transiently transfected U2-OS and/or CHO cells (supplemental Figs. 8 –12). Colocalization between the mCherry-can-
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Mannose 6-Phosphate Glycoproteome
FIG. 4. Enrichment factors for assigned proteins in affinity column eluates. Panels A and B, log2 of the ratio of spectral counts in the
combined Man6P and Glc6P/Man eluates for all experiments combined is plotted with bars representing the upper and lower 95% confidence
indices. Plots represent the following: blue error bars, known Man6P glycoproteins; green error bars, other proteins that are significantly (q ⱕ
0.05) enriched in the Man6P eluate; gray error bars, other proteins that cannot be classified (q ⬎ 0.05); and red error bars, other proteins that
are significantly (q ⱕ 0.05) depleted in the specific eluate. Fold-enrichments that are greater than 16 or less than 1/16 are arbitrarily assigned
to be 16 (a log2 value of 4) or 1/16 (a log2 value of ⫺4), respectively. Data were filtered for proteins with total spectral counts of ⱖ20, and those
of non-murine origin are not shown. Panel A, proteins identified in preparations of purified Man6P glycoproteins from mouse. Panel B, proteins
identified in preparations of purified Man6P glycoproteins from rat (previously reported in Ref. 3). Panel C, correlation of enrichment factors
(proportion of spectral counts in Man6P eluate of the total spectral counts in Glc6P/Man and Man6P eluates) for proteins identified in both
mouse and rat. Red, known Man6P glycoproteins; black other proteins.
didate fusion and with fluorescent-labeled NPC2 or dextran
lysosomal marker was quantified (supplemental Table 11). For
APCS and PLD3, we observe a punctate cytoplasmic distribution of the fusion proteins with extensive colocalization with
the lysosomal marker. When all experiments are considered
together, we obtained a thresholded Mander’s colocalization
coefficient between the lysosomal markers and APCS and
1812
PLD3 of 0.81 and 0.83, respectively. For RNASE6, we also
find significant colocalization with the lysosomal marker (average thresholded Mander’s colocalization coefficient, 0.66),
but there is additional cytoplasmic staining. For LPO, diffuse
reticular cytoplasmic staining predominates, but careful inspection does reveal additional punctate cytoplasmic staining
that colocalizes with the lysosomal marker (average thresh-
Molecular & Cellular Proteomics 12.7
Mannose 6-Phosphate Glycoproteome
TABLE II
Comparison of common protein assignments from mice and rat
Note that there are three proteins that were not classified as lysosomal in the original analysis of rat tissues (Il4i1, Ctso, and Scpep1 (3))
that we now consider to be validated lysosomal proteins. Data were
filtered for proteins with total spectral counts of ⱖ20.
Specificity, rat
Classification
Specificity,
mouse
Enriched
Ambiguous
Depleted
Known
Man6P
Other
Enriched
Depleted
Enriched
Ambiguous
Depleted
62
0
41
4
1
1
0
5
2
10
0
1
7
7
68
TABLE III
Classification of proteins that are significantly elevated in Man6P versus
Glc6P/Man eluates from combined analysis of all rodent samples
Protein type
Total
Enzyme, nonhydrolase
Glycosyltransferase
Hydrolase
Protease inhibitor
Ribosomal protein
Small leucine-rich proteoglycan
Other
Total
30
15
44
20
9
6
70
194
olded Mander’s colocalization coefficient, 0.48). In cells expressing low amounts of the LPO fusion construct, the
mCherry signal clearly colocalizes with the lysosomal marker
(for example, see Fig. 5, panel LPO, cell b). These results
provide convincing evidence that intracellular APCS and
PLD3 primarily reside within the lysosome and suggest that
some of the intracellular LPO and RNASE6 also reside within
this organelle.
CONCLUSIONS
This study exploits our earlier discovery that the Man6P
lysosomal recognition signal is removed within the lysosome
by the lysosomal phosphatase, ACP5. We demonstrate that
preparations of affinity-purified Man6P glycoproteins from
mice lacking ACP5 have significantly lower levels of nonlysosomal contaminants than preparations from wild-type
mice. However, we find that the presence of the additional
contaminants associated with the sample purified from
wild-type animals has no adverse effect on the identification
of known Man6P glycoproteins and presumably lysosomal
candidates. This is likely because the purified samples from
both mice genotypes are of relatively low complexity, and
thus analytical capacity is not a limiting factor with the
specific instrumentation and workflow employed to achieve
the goals of this study.
Spectral counts of most known Man6P glycoproteins were
relatively increased in preparations of Man6P glycoproteins
from Acp5⫺/⫺ liver compared with wild type. Among the ex-
Molecular & Cellular Proteomics 12.7
FIG. 5. Subcellular localization of lysosomal candidate-mCherry
fusion proteins. Stably transfected CHO cells expressing fusion proteins between mCherry and lysosomal candidates APCS, LPO, PLD3,
and RNASE6 were treated for 24 h with AlexaFluor-488-labeled
NPC2, which is endocytosed and delivered to the lysosome. Left
column, visualization of mCherry fluorescence; middle column, visualization of AlexaFluor-488-labeled NPC2; right column, merged images showing colocalization (yellow) between the fusion proteins (red)
and NPC2 (green). Cells indicated to their right with “a” or “b” were
selected for colocalization analyses (supplemental Table 11).
ceptions were ␤-glucuronidase, prosaposin, and several
cathepsins (Fig. 3 and supplemental Table 2). It is not clear
why the Man6P forms of these proteins are not elevated in
liver, but it is possible that they are preferential substrates for
an alternative ACP5-independent pathway for the removal of
Man6P. For example, there is evidence that lysosomal acid
phosphatase (ACP2) may play a role in the dephosphorylation
of lysosomal proteins (21) or alternatively, Man6P could be
lost as a consequence of deglycosylation. The fact that the
loss of ACP5 does not in result in the complete retention of
Man6P in liver suggests that such a pathway exists. It is worth
noting that in an earlier study (5), we found that the loss of
ACP5 did not result in the retention of Man6P on ␤-glucuronidase to the same extent as it does for ␤-galactosidase and
␤-hexosaminidase, providing support for the possibility that it
may be partially dephosphorylated by an ACP5-independent
pathway.
Candidate lysosomal proteins arising from this study are
shown in Table III and supplemental Table 10, and they fall
into several broad classes. We identified numerous protease
inhibitors, and it is likely that many of these represent false
1813
Mannose 6-Phosphate Glycoproteome
positives that are purified in association with lysosomal proteases. We also identified a number of endoplasmic reticulum
(ER) glycosyltransferases. It is possible that these may be
purified with lysosomal glycoproteins if they possess lectinlike properties. However, it is worth noting that for some (e.g.
B3GNT1 and POFUT2), we previously demonstrated that they
do contain Man6P (22). This suggests that some or all of this
class of specifically purified proteins may actually represent
true Man6P glycoproteins. The biological significance of the
Man6P lysosomal targeting modification on ER glycosyltransferases is unclear. It may be that these proteins are low affinity
substrates for the phosphotransferase responsible for the
addition of Man6P, and they receive the modification as a
result of prolonged exposure to the pool of phosphotransferase residing in the ER (23).
Six small leucine-rich repeat proteoglycans (SLRRPs) were
specifically purified. SLRRPs are a class of proteins containing repetitive leucine sequences and various classes of glycosaminoglycan side chains, including keratin sulfate, chondroitin sulfate, dermatan sulfate, and heparin sulfate chains.
Originally recognized for their structural properties, there is
increasing evidence that proteoglycans such as SLRRPs play
a role in cellular signaling (24). Again, it is not clear whether
their specific isolation here reflects the presence of Man6P
or interaction with true Man6P glycoproteins (as outlined
above). There is evidence that proteoglycans of the serglycin type may interact with the CI-MPR in competition with
Man6P-containing ligands, suggesting that this may be a
direct interaction (25). In addition, SLRRPs contain O-linked
glycans, and there is evidence that another protein (␣dystroglycan) contains similar structures with phosphorylated O-mannosyl residues that may possibly interact with
the CI-MPR (26).
The most numerous of the specifically isolated proteins
were catabolic hydrolases, and given that the majority of
lysosomal Man6P glycoproteins also fall within this class,
we have examined these proteins in more detail (Table IV).
The majority of the enriched hydrolases were proteases/
peptidases with a large number of serine proteases identified, including eight members of the family of kallikreinrelated peptidases. Many of the enriched hydrolases have a
relatively limited cellular distribution; for example, we find a
number of mast cell-specific proteases and eosinophil-specific ribonucleases, suggesting that some cell types may
have unique lysosomal proteomes reflecting specialized
function.
A number of candidates were identified with primary sequence and/or functional similarities to known lysosomal proteins. Most of these candidates (e.g. SMPDL3A, PLBD1,
ARSK, and RNASE6) have been discussed previously (2), but
one notable lysosomal candidate is identified in this study.
␤-Galactosidase 1-like (GLB1L) was present at relatively low
levels based on total spectral counts but was highly enriched
in the Man6P eluate. This protein has significant sequence
1814
similarity to ␤-galactosidase 1 (human GLB1 and GLB1L are
55% identical and 67% similar), and although it is likely to be
a glycosylhydrolase, its precise catalytic function remains to
be determined. It is interesting to note that of the tissues
analyzed in the Acp5⫺/⫺ mice, expression of GLB1L was almost
completely restricted to the reproductive system, with 34 of the
42 total spectral counts derived from testis and epididymis and
four derived from ovary. Examination of the EST profile
(www.ncbi.nlm.nih.gov/UniGene/ESTProfileViewer.cgi?
uglist ⫽ Hs.181173) for the human ortholog also shows
highest relative expression levels in testis. These results
suggest that this protein may play a specialized role in
reproduction.
We investigated the subcellular localization of four candidates arising from this study by expressing fluorescenttagged fusion constructs and found evidence for lysosomal
localization for each. PLD3 (phospholipase D family, member
3) was previously identified as a glycosylated, endoplasmic
reticulum-resident transmembrane protein (27). A recent
study showing that PLD3 colocalizes with lysosomal protein
LAMP1, and that its expression is mediated by a transcription
factor that regulates lysosomal biogenesis and function (28),
is consistent with a lysosomal distribution. LPO is found in
secretory fluid such as milk and saliva and plays an important
role in host defense. LPO is a member of the heme family of
peroxidases, which includes myeloperoxidase, a component
of neutrophil secretory granules that contains Man6P (29) that
may be important for segregation and intracellular transport.
Immunohistochemical analysis from the Human Protein Atlas
Project (30) indicates some cytoplasmic staining for LPO that
may be consistent with lysosomal localization. However, we
also observed diffuse cytoplasmic staining for LPO that may
reflect ER localization. Ribonuclease k6 (RNASE6) is a ubiquitously expressed ribonuclease that has also been postulated to play a role in host defense (31). To date, the only
ribonuclease that has been localized to the lysosome is ribonuclease T2 (32). Our results suggest that RNASE6 represents
an additional lysosomal, Man6P-containing ribonuclease, and
the first of the RNase A family to be shown to reside within this
organelle.
A lysosomal localization for PLD3, LPO, and RNASE6 is
consistent with their predicted enzymatic activities and the
presence of similar enzymes within the organelle. However,
one intriguing result of this study was the lysosomal localization of serum APCS, a circulatory glycoprotein that binds
␤-amyloid peptide (33) and that is a component of human
amyloid deposits (34). Given the intriguing connections between the lysosomal system and Alzheimer disease (35), the
observation that APCS can reside within the lysosome may
have significant implications for disease.
Analysis of Man6P glycoproteins in this study and elsewhere has resulted in a significant number (⬃50 –100) of new
proteins that may potentially reside within the lumen of the
lysosome, and systematic validation of their localization will
Molecular & Cellular Proteomics 12.7
Mannose 6-Phosphate Glycoproteome
TABLE IV
Lysosomal hydrolase candidates
Data were obtained from mouse and rat (all experiments, supplemental Table 1) and proteins with known or predicted hydrolase activity are
shown (supplemental Table 10). Gene symbols correspond to mouse equivalents except for Mcpt8l3 which is present only in rat. Blank entries
indicate respective protein not found.
Specificity of purification
Gene symbol
Protein description
2210010C04Rik
Adam28
Amy2a1
Arsk
Bche
Btd
Ces1c
Ces1e
Ces2g
Ces5a
Cma1
Cp
Cpa2
Cpa3
Cpb2
Cym
Ear1
Ear2
Ear6
Erap1
Fuca2
Glb1l
Klk1b1
Klk1b11
Klk1b16
Klk1b27
Klk1b3
Klk1b4
Klk1b5
Klk1b9
Mcpt1
Mcpt2
Mcpt8
Mcpt8l3
Minpp1
Ngly1
Plbd1
Pld3
Pnliprp1
Prtn3
Rnase6
Smpdl3a
Tpsab1
Vnn3
Trypsin-3
A disintegrin and metallopeptidase domain 28
Pancreatic ␣-amylase
Arylsulfatase K
Butyrylcholinesterase
Biotinidase
Carboxylesterase 1C
Carboxylesterase 1E
Carboxylesterase 2G
Carboxylesterase 5A
Chymase 1, mast cell
Ceruloplasmin
Carboxypeptidase A2
Carboxypeptidase A3 mast cell
Carboxypeptidase B2 (plasma)
Embryonic pepsinogen
Eosinophil-associated ribonuclease A family
Eosinophil-associated ribonuclease A family
Eosinophil-associated ribonuclease A family
Endoplasmic reticulum aminopeptidase 1
Fucosidase ␣-L-2
Galactosidase ␤1-like
Kallikrein 1-related peptidase b1
Kallikrein 1-related peptidase b11
Kallikrein 1-related peptidase b16
Kallikrein 1-related peptidase b27
Kallikrein 1-related peptidase b3
Kallikrein 1-related peptidase b4
Kallikrein 1-related peptidase b5
Kallikrein 1-related peptidase b9
Mast cell protease 1
Mast cell protease 2
Mast cell protease 8
Mast cell protease 8-like 3
Multiple inositol polyphosphate histidine phosphatase
N-Glycanase 1
Phospholipase B domain containing 1
Phospholipase D family member 3
Pancreatic lipase-related protein 1
Proteinase 3
Ribonuclease A family
Sphingomyelin phosphodiesterase acid-like 3A
Tryptase ␣/␤1
Vanin 3
be the next major step in defining this part of the lysosomal
proteome. In this study, we have used fluorescent-tagged
expressed proteins to demonstrate colocalization with two
lysosomal markers (fluorescent NPC2 and dextran) in two
different cell types (CHO and U2-OS), and this approach does
demonstrate that the respective candidates can be delivered
to this organelle. Two of the candidate fusions (PLD3 and
APCS) appear to predominantly localize to the lysosome, but
for LPO and to a lesser extent, RNASE6, we also observe
nonlysosomal cytoplasmic staining. The physiological relevance of nonlysosomal staining in the latter cases is not clear.
It can be argued that the approach of expression of fluores-
Molecular & Cellular Proteomics 12.7
Mouse
Depleted
Elevated
Elevated
Elevated
Elevated
Ambiguous
Elevated
Elevated
Elevated
Elevated
Elevated
Elevated
Elevated
Elevated
Elevated
Elevated
Elevated
Elevated
Elevated
Elevated
Elevated
Elevated
Elevated
Elevated
Elevated
Elevated
Elevated
Elevated
Elevated
Elevated
Elevated
Elevated
Elevated
Elevated
Rat
Elevated
Elevated
Elevated
Elevated
Elevated
Elevated
Elevated
Elevated
Elevated
Elevated
Elevated
Elevated
Elevated
Elevated
Elevated
Elevated
Elevated
Elevated
Elevated
Elevated
Elevated
Elevated
Elevated
Elevated
Elevated
cent fusion proteins, although a mainstay of cell biology, is
perturbing, and thus results may not necessarily reflect the
distribution of the native endogenous protein. For example,
the C-terminal mCherry fusion could interfere with normal
protein folding or glycosylation, and this may in turn disrupt
normal intracellular targeting. Alternatively, overexpression
may interfere with normal targeting pathways, resulting in
abnormal subcellular location, e.g. an accumulation within the
ER. However, it is also possible that LPO and RNASE6 normally have multiple cellular localizations. An example of such
a protein is superoxide dismutase 1 (36). Thus, in future
studies it will be important to determine the proportion of a
1815
Mannose 6-Phosphate Glycoproteome
given protein that localizes to the lysosome, and this will be
key in interpreting lysosomal residence. For example, for a
given protein that is predominantly secreted with a small
proportion retained intracellularly within the lysosome, interpretation of the lysosomal residence demonstrated by morphological methods is not as straightforward as that of a
classical lysosomal protein that is primarily targeted to the
organelle. Similar considerations will apply to proteins that
have multiple intracellular locations, including the lysosome
and nonlysosomal proteins undergoing lysosomal degradation. Future analyses applicable to lysosomal candidates, including analysis of cellular retention and targeted mass spectrometry to investigate subcellular distribution in differential
centrifugation fractions (36), will provide useful information in
such cases.
Supporting Information—Raw mass spectrometry data files
will be made available in the Tranche public repository. Supporting information in the form of an Excel workbook is provided with information for protein assignment and statistical
analyses.
Acknowledgments—We thank Caifeng Zhao and Meiqian Qian for
their excellent assistance with the mass spectrometry. We would also
like to thank Dr. Zhiping Pang and the Child Health Institute of New
Jersey (supported by the Robert Wood Johnson Foundation) for
assistance with confocal microscopy.
* This work was supported, in whole or in part, by National Institutes of Health Grants DK054317 and S10RR017992 (to P.L).
□
S 兩This article contains supplemental material.
¶ To whom correspondence may be addressed: Center for Advanced Biotechnology and Medicine, 679 Hoes Ln., Piscataway, NJ
08854. Tel.: 732-235-5313; Fax: 732-235-4466; E-mail: sleat@cabm.
rutgers.edu (to D.E.S.) or Center for Advanced Biotechnology and
Medicine, 679 Hoes Ln., Piscataway, NJ 08854. Tel.: 732-235-5032;
Fax: 732-235-4466; E-mail: [email protected]. (to P.L.).
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.
21.
22.
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