Journal of Leukocyte Biology BioLegend: Treg, Th17, Stem Cell
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Originally published online as doi:10.1189/jlb.0806520 on January 8, 2007

Published online before print January 8, 2007
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
jlb.0806520v1
81/4/1149    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Lemansky, P.
Right arrow Articles by Hasilik, A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Lemansky, P.
Right arrow Articles by Hasilik, A.
(Journal of Leukocyte Biology. 2007;81:1149-1158.)
© 2007 by Society for Leukocyte Biology

The cation-independent mannose 6-phosphate receptor is involved in lysosomal delivery of serglycin

Peter Lemansky1, Ines Fester, Eva Smolenova, Christoph Uhländer and Andrej Hasilik

Institut für Physiologische Chemie, Philipps-Universität Marburg, Marburg, Germany

1 Correspondence: Institut für Physiologische Chemie, Karl-von-Frisch-Str. 1, 35043 Marburg/Lahn, Germany. E-mail: lemansky{at}staff.uni-marburg.de


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
To clarify the sorting mechanism of the lysosomal/granular proteoglycan serglycin, we treated human promonocytic U937 cells with p-nitrophenyl-ß-D-xyloside (PNP-xyl) and cycloheximide. In the absence of protein synthesis, the carbohydrate moiety of serglycin was synthesized as PNP-xyl-chondroitin sulfate (CS), and most of it was delivered to lysosomes and degraded. Further, an augmented lysosomal targeting of serglycin in the presence of tunicamycin suggested that a sorting/lectin receptor with multiple specificity was involved with an increased capacity for serglycin in the absence of N-glycosylation. Correspondingly, the cation-independent mannose 6-phosphate receptor (CI-MPR) and sortilin were observed to bind to immobilized CS. These receptors were eluted in the presence of 200–400 mM and 100–250 mM NaCl, respectively. After treating the cells with a cross-linking reagent, a portion of the sulfated proteoglycan was coimmunoprecipitated with the CI-MPR but not with sortilin. In the presence of phorbol ester, lysosomal targeting of serglycin and to a lesser extent, of cathepsin D was inhibited. We conclude that the CI-MPR participates in lysosomal and granular targeting of serglycin and basic proteins such as lysozyme associated with the proteoglycan in hematopoietic cells.

Key Words: chondroitin sulfate • lysozyme • phorbol ester • sortilin


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Here, we report about a hitherto unknown function of the cation-independent mannose 6-phosphate receptor (CI-MPR), which is the lysosomal delivery of serglycin, a soluble proteoglycan found in many different hematopoietic cell types. In the past few years, it became increasingly clear that serglycin plays an important role in the biogenesis of lysosomes and lysosome-related organelles of hematopoietic cells (reviewed in ref. [1 ]). Examples are the secretory granules in mast cells [2 , 3 ], the azurophilic granules in HL-60 cells [4 ], or the secretory granules in CTL [5 ]. These studies showed invariably that many of the bactericidal proteins possess a high isoelectric point and associate with serglycin through electrostatic interactions. Formation of these complexes was a prerequisite for the normal biogenesis of the respective organelles [3 , 5 ]. Many of these organelles are acidic and contain lysosomal enzymes, indicating a trafficking of mannose 6-phosphate (M6P)-containing lysosomal enzymes to these granules. This may implicate an involvment of MPRs in their biogenesis. Hitherto unexplained is the granular targeting of bactericidal enzymes and polypeptides and their complexes with serglycin.

Although the protein portion of serglycin is the same in every cell type, its glycosaminoglycan (GAG) portion can vary considerably. In mast cells, it consists of heparin, heparan sulfate, or chondroitin sulfate (CS) chains, whereas in U937 cells, it consists entirely of CS chains. Experiments dealing with the sorting of serglycin in rat acinar pancreatic cells showed that the GAG portion of serglycin was necessary for its correct targeting [6 ]. This result does not necessarily imply that targeting of serglycin in other cell types relies on the GAG portion of serglycin too. We therefore examined whether p-nitrophenyl-ß-D-xyloside (PNP-xyl)-primed CS chains were targeted correctly to lysosomes in U937 cells after blocking protein biosynthesis for 27 h. We found that lysosomal delivery of CS continued unimpeded in the presence of cycloheximide, indicating that it was independent of an ongoing protein biosynthesis. This suggested that sorting was independent of proteins or was carried out by a recycling receptor system.

To date, there are several receptor families known to mediate lysosomal sorting of soluble ligands. Two of them are the MPRs [7 ] and the Vps10p receptor family, a representative of which is sortilin [8 ]. All members of this family possess a highly conserved Vps10 domain and a cytoplasmic tail resembling that of the MPRs [9 ]. Sortilin has two positively charged clusters of arginines and lysines within its Vps10 domain consisting of R644LRK and K737KK, whereas the CI-MPR has five such clusters (K481KR, K576KIK, R1116KR, R1466KK, and K1579RLR), which may be able to facilitate binding to negatively charged GAG chains. Sortilin mediates cellular trafficking of several unrelated ligands, such as neurotensin [10 ], lipoprotein lipase [11 ], brain-derived neurotrophic factor [12 ], Glut4 [13 ], and sphingolipid activator proteins [14 ]. In addition to sortilin, related receptors have been described, which are also expressed primarily in brain-derived tissues, such as sorLA [15 ] and sorCS 1-3 [16 17 18 ].

In our study, we used the human promonocytic U937 cell line to examine whether the CI-MPR, the cation-dependent CD-MPR, and sortilin play a role in lysosomal targeting of serglycin. In one experiment, the analysis of the CI-MPR was extended to the human promyelocytic HL-60 cell line. Through confocal microscopy, we found that the CI-MPR colocalized with serglycin to some extent in U937 cells. The ability of the MPRs and sortilin to bind to serglycin was examined by CS-Sepharose chromatography. The membrane-permeable, sulfhydryl-cleavable cross-linker dithio-bis(succinimidylpropionate; DSP) was then used to covalently cross-link macromolecular complexes within U937 cells after labeling serglycin with [35S]sulfate. Formation of serglycin/receptor complexes was monitored by immunoprecipitation. Furthermore, we treated U937 cells with tunicamycin and 4-ß-PMA, agents that greatly influence lysosomal delivery of serglycin, and examined their effect on the trafficking of lysosomal receptors to explore possible interactions.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Materials
CS, a mixture of 70% chondroitin-4-sulfate and 30% chondroitin-6-sulfate, was purchased from Sigma (Deisenhofen, Germany) and attached to cyanogen bromide (CNBr)-activated Sepharose Cl–4B, according to the manufacturer’s instructions (Amersham Pharmacia, Freiburg, Germany). DSP and IodobeadsTM were from Perbio Science (Bonn, Germany). Tunicamycin and PansorbinTM, a cell-wall preparation of Staphylococcus aureus, was obtained from Calbiochem (Merck Biosciences, Schwalbach, Germany). Calcitriol (1,25-dihydroxycholecalciferol), PMA, cycloheximide, and all other reagents were from Sigma.

Cy3-conjugated, affinity-purified goat antirabbit IgGs or Cy2-conjugated, affinity-purified goat antimouse IgGs were from Dianova (Hamburg, Germany). The polyclonal goat antiserum directed against part of the C terminus of human serglycin (D-19) was obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA), whereas the rabbit antiserum against recombinant human serglycin, lacking carbohydrate, was a gift from Dr. Carsten U. Niemann (Rigshospitalet, Copenhagen, Denmark). The mouse mAb against human cathepsin D (CD) was a gift from Dr. Stephen Weitz (Fibro Inc., South San Francisco, CA, USA). The rabbit antibody against human cathepsin D was raised in our own laboratory, and the rabbit antisera, specific for human lysozyme [19 ], myeloperoxidase (MPO) [4 ], and CI-MPR [20 ], were those described earlier. The rabbit antihuman sortilin antiserum was a gift from Dr. Claus M. Petersen (Aarhuis University, Denmark). The goat antihuman CI-MPR antiserum was from Dr. Kurt von Figura (University of Göttingen, Germany).

Culturing and metabolic labeling of cells
The human promonocytic U937 or promyelocytic HL-60 cell line was cultured in RPMI medium, containing 10% heat-inactivated FCS (both from Gibco-BRL, Eggenstein, Germany) and supplemented with 100 units/ml penicillin and 100 µg/ml streptomycin. In some experiments, stimulated expression of lysozyme and lysosomal enzymes was obtained by culturing U937 cells in the presence of 0.1 mM calcitriol for at least 3 days.

Metabolic labeling was performed with [35S]sulfate (92.5 MBq/ml) or [35S]Met-label (3.7 MBq/ml) purchased from Hartmann Analytic (Braunschweig, Germany) in sulfate- or L-methionine/L-cysteine-deficient RPMI medium, respectively. The labeling medium contained antibiotics and 10% heat-inactivated FCS, which was dialyzed against 0.9% NaCl. Before addition of the radioactive tracer, U937 cells were washed three times in deficient RPMI medium and kept in this medium for 1–3 h. PMA was dissolved in DMSO at a concentration of 0.1 mM before application to the cells (final concentration, 0.1 or 0.2 µM). Control cells were incubated with 0.1 or 0.2% (v/v) DMSO.

Isolation and iodination of membrane-associated proteins from U937 cells
U937 cells were extracted with 50 mM Tris/Cl, pH 7.4/100 mM NaCl/0.1% saponin/10% FCS/5 mM iodoacetamide (IAA)/5 mM EDTA/2 mM PMSF, for 10 min at room temperature (RT). Extracts were then centrifuged for 1 h at 100,000 g, 4°C, and re-extracted and recentrifuged twice with the same buffer. Finally, the cells were extracted with 50 mM Tris/Cl, pH 7.4/100 mM NaCl/0.5% Triton X-100 (TX-100)/5 mM IAA/2 mM PMSF, for 10 min at RT and centrifuged for 10 min at 20,000 g, 4°C. The saponin extracts contained soluble proteins, whereas the TX-100 extract contained the membrane proteins of U937 cells. The latter were iodinated for 15 min with IodobeadsTM and 16 MBq [125I]NaI (630 GBq/mg, Hartmann Analytic) at RT, following the instructions of the manufacturer (Perbio Science). Free radioactive iodide was removed on a Sephadex G-10 column equilibrated with 50 mM Tris/Cl, pH 7.4/100 mM NaCl/0.5% TX-100.

Gel electrophoresis-related techniques and DEAE or TCA precipitation of [35S]sulfate-labeled proteoglycans
SDS-PAGE was performed according to Laemmli [21 ], followed by fluorography [22 ]. Gels containing 125I-labeled proteins were subjected to autoradiography with the help of intensifier screens.

Quantification of [35S]sulfate-labeled proteoglycans was performed by incubating aliquots of [35S]sulfate-labeled secretions or cell lysates with 20 µl DEAE-Sephacel for 30 min at 4°C. After a 1-min centrifugation at 14,000 g, pellets were washed three times with 0.8 ml ice-cold 50 mM Tris/Cl, pH 7.4/145 mM NaCl/0.5% TX-100, and dissolved in 50 µl-reducing Laemmli sample buffer containing 2.4% SDS, boiled for 5 min at 95°C, and subjected to liquid scintillation counting. In some experiments, one-half of each sample was analyzed additionally by SDS-PAGE and fluorography. In other experiments, quantification of [35S]sulfate-labeled serglycin was performed by TCA precipitation as described earlier [4 ].

Affinity chromatography with immobilized CS or serglycin antibodies and other techniques
CS-Sepharose Cl–4B columns (5x8 mm) were equilibrated with 20 mM Tris-HCl, pH 7.4/30 mM NaCl/0.5% TX-100/5 mg/ml BSA (Buffer A), and allowed to bind 125I-labeled membrane proteins from U937 cells for 10 min at RT. The columns were then washed extensively with Buffer A and Buffer A without BSA. Elution was performed with application of Buffer A without BSA, containing increasing concentrations of NaCl.

Serglycin-specific antibodies (D-19) were attached to CNBr-activated Sepharose Cl–4B. D-19 minicolumns (5x8 mm) were equilibrated with 50 mM Tris/Cl, pH 7.4/150 mM NaCl/0.1% BSA/0.5% Triton X-100 (Buffer B with BSA). Sample (50 µl) was then applied and incubated for 2 h at RT with the column matrix. The column was then washed with Buffer B containing 150 mM, 650 mM, 1.15 M, and 2.15 M NaCl, briefly washed with Buffer B, and eluted with 0.1 M Na2CO3/0.5% TX-100. Fractions were neutralized immediately with HCl and analyzed by liquid scintillation counting and SDS-PAGE.

Sucrose density gradient centrifugation, immunoprecipitations, and cross-linking of proteins in U937 cells with DSP were performed as described earlier [4 ], except that two additional washing steps for immunoprecipitates were included. These washing steps were performed with 2 M KCl added to the initial washing buffer.

Immunocytochemistry and confocal microscopy
U937 cells were attached to polylysine-coated coverslips, fixed with 5% formaldehyde for 30 min, permeabilized in PBS/0.3% TX-100 for 3 min, and blocked with PBS/3% BSA for 30 min. The primary antibodies, rabbit antiserglycin, mouse anti-CD, and mouse anti-CI-MPR, were dissolved in PBS and added overnight at 4°C. After washing, Cy2- and Cy3-labeled secondary antibodies, diluted 1:250 or 1:1000, respectively, in PBS, were applied for 45 min at RT. The cells were then washed with PBS and mounted on microscopic slides using 50% MowiolTM. The specimen was analyzed at RT using a Zeiss Axioplan 2 confocal microscope equipped with Plan-Neofluar lenses. Pictures were taken by an Axiocam MRm Zeiss camera using the Axioplan 2 imaging software for 3D reconstruction.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Lysosomal targeting of CS chains in the absence of protein biosynthesis
U937 cells were pretreated with 1 mM PNP-xyl for 3 days, with or without 0.1 mg/ml cycloheximide for 27 h prior to labeling with [35S]sulfate for 15 min. The chase period was initiated by the addition of medium containing 10 mM MgSO4.

After the indicated times (Fig. 1 ), cells were washed and lysed, and aliquots of cell lysates and media were incubated with DEAE-Sephacel beads to collect [35S]sulfate-labeled proteoglycans or PNP-xyl-primed CS chains. The [35S]sulfate-labeled material was then analyzed by SDS-PAGE (Fig. 1A) and by liquid scintillation counting (Fig. 1B) .


Figure 1
View larger version (60K):
[in this window]
[in a new window]

  		Figure 1. Lysosomal delivery of PNP-xyl-primed CS chains in the absence of protein biosynthesis. U937 cells were pretreated with 1 mM PNP-xyl for 73 h. Half of the cells were additionally pretreated with 0.1 mg/ml cycloheximide for 27 h. Cells were then labeled with [35S]sulfate for 15 min and chased for up to 230 min in the continued presence or absence of the drugs. [35S]Sulfate-labeled material was collected by DEAE-Sephacel beads, boiled in reducing sample buffer, and analyzed by SDS-PAGE and fluorography (A) or liquid scintillation counting (B). Solid lines, {diamondsuit}, Treatment with PNP-xyl. Dashed lines, {triangleup}, treatment with PNP-xyl and cycloheximide. The {gamma}-setting of the scanner acquiring the image was set to allow a clear, optimal presentation of bands in this and all following images.

 
In the presence of PNP-xyl, U937 cells synthesized CS chains bound to serglycin or to PNP-xyl. The latter were visible as a "laddering" material, migrating below the 46-kDa standard. Both types of CS chains were partially secreted and partially degraded with increasing time. For serglycin, degradation was shown to be executed within endosomes and/or lysosomes [19 ]. A few [35S]sulfate-labeled polypeptides were visible as faintly labeled bands as compared with the massive [35S]sulfate label incorporated into proteoglycans and GAGs. In the presence of PNP-xyl and cycloheximide, [35S]sulfate-labeled serglycin and polypeptides vanished. Only the [35S]sulfate-labeled CS chains primed by PNP-xyl were readily visible with residual traces of [35S]sulfate-labeled serglycin. CS chains primed by PNP-xyl were secreted to a small extent, but most of them were subject to intracellular degradation. This indicates that PNP-xyl-primed CS chains were delivered to the lysosomal compartment in the absence of a serglycin polypeptide backbone. Although lysosomal targeting of CS did not require the serglycin moiety, it is not ruled out that this moiety contributes to this process. Furthermore, transport of CS chains was independent of an ongoing protein biosynthesis, suggesting the involvement of one or more long-lived sorting receptors.

CS affinity chromatography of sortilin and the MPRs
In this experiment, we measured binding of sortilin and the MPRs to CS. To this end, 125I-labeled membrane proteins from U937 cells were applied to a CS-sepharose column, which was then washed extensively and eluted with an increasing NaCl stepgradient.

Sortilin and the MPRs were immunoprecipitated from all fractions and from the flow-through or starting material, respectively, and analyzed by SDS-PAGE and autoradiography (Fig. 2 ).


Figure 2
View larger version (41K):
[in this window]
[in a new window]

  		Figure 2. CS affinity chromatography of 125I-labeled CI-MPR and sortilin. 125I-Labeled membrane proteins from U937 cells were passed over a CS-Sepharose Cl–4B column. After washing the column thoroughly, bound polypeptides were eluted with increasing NaCl concentrations. The CI-MPR and sortilin were immunoprecipitated from 11% of the flow-through (FT) or 7.5% of the starting material (SM), respectively, and from the resulting fractions and visualized by SDS-PAGE and autoradiography. Binding of the CI-MPR to CS-sepharose required higher salt concentrations for elution (100–450 mM NaCl) as compared with sortilin, which was eluted by 100–250 mM NaCl.

 
125I-labeled sortilin, immunoprecipitated from U937 cells, migrated slightly faster than the 97.4-kDa standard in SDS-PAGE analyses and was eluted from the CS column at NaCl concentrations ranging from 150 to 250 mM NaCl. CS affinity chromatography in the presence of 2 mM CaCl2 and pH 6.8 resulted in elution of sortilin with 200–300 mM NaCl (not shown), indicating that binding of sortilin to CS is enhanced moderately by approaching the conditions of the trans-Golgi network (TGN). Binding of the CI-MPR to CS-sepharose was even better, as it required higher salt concentrations for elution (100–450 mM NaCl).

Binding of the CD-MPR to CS-sepharose in the presence of 2 mM CaCl2 and pH 6.8 was not observed.

Colocalization of CD and the CI-MPR with serglycin in U937 cells and cross-linking of serglycin with different antigens
We examined by immunocytochemistry and confocal microscopy the subcellular distribution of CD, the CI-MPR, and serglycin in U937 cells. CD is a typical lysosomal proteinase [23 ] and is colocalized with serglycin, only to a small degree in agreement with a rapid lysosomal degradation of serglycin [19 ]. On its way to lysosomes, serglycin colocalized with the CI-MPR to a large extent, although the occurrence of uniquely green- or red-labeled vesicles indicates that colocalization is incomplete.

Next, we examined whether the proteoglycan(s) from [35S]sulfate-labeled U937 cells were indeed serglycin. Therefore, cell extract from these cells was precipitated by TCA or passed over a column with immobilized D-19 serglycin antibodies. Radioactivity contained in these samples was quantified by liquid scintillation counting and analyzed by SDS-PAGE and fluorography (Fig. 3B , left panel). Sixty-five percent of the TCA-precipitable, [35S]sulfate-labeled material was recovered from the D-19 column. This portion increased to 99.6% when PMA-induced secretions were analyzed in the same way (not shown). The latter material contains virtually all of the newly synthesized proteoglycan (see Fig. 4 ) and most important, as intact molecules, as they do not encounter activated lysosomal proteinases. These results indicate that the overwhelming majority of [35S]sulfate-labeled proteoglycans does possess a serglycin backbone in U937 cells.


Figure 3
View larger version (27K):
[in this window]
[in a new window]

  		Figure 3. Colocalization of serglycin with CI-MPR and CD and cross-linking with DSP. (A) U937 cells were fixed with formaldehyde, permeabilized, and processed for immunocytochemistry of CD or the CI-MPR plus serglycin, followed by confocal microscopy. Original magnification was 1000-fold using immersion oil Immersol 518F. There was much colocalization of the CI-MPR and little colocalization of CD with serglycin. Original bar, 2.5 µm. DAPI, 4', 6-Diamidino-2-phenylindole. (B) Cell extract from U937 cells labeled with [35S]sulfate for 10 min was precipitated by TCA or passed over a D-19 column with immobilized serglycin antibodies. Aliquots of the TCA-precipitable material and the alkaline eluate of the column were analyzed by liquid scintillation counting and SDS-PAGE followed by fluorography (Fig. 3B , left panel). Sixty-five percent of the TCA-precipitable, [35S]sulfate-labeled material was recovered from the D-19 column, whereas recovery increased to 99.6% when PMA-induced secretions were analyzed in the same way (not shown). U937 cells were labeled with [35S]sulfate for 30 min and washed, and one-half of the cells was treated with 1 mM DSP for 10 min at 37°C, whereas the other half was mock-incubated for 10 min at 37°C. Immunoprecipitates were analyzed by liquid scintillation counting (one-third of the sample) and by reducing SDS-PAGE and fluorography (two-thirds of the sample; Fig. 3B , right panel). Serglycin showed strong cross-linking to lysozyme (Lys), medium cross-linking to the CI-MPR, and barely any cross-linking to sortilin (Sor). The position of sulfated serglycin (Sergl.) and CI-MPR is indicated by bars. Cross-linking of the CI-MPR to serglycin caused a 2.7-fold increase in the number of immunoprecipitable radioactivity compared with the noncross-linked sample, as determined by liquid scintillation counting. Co, Control. (C) U937 cells were labeled with [35S]sulfate for 10 min, washed, and treated with 1 mM DSP for 10 min at 37°C. Immunoprecipitation was performed as above with nonimmune serum (Co) and lysozyme, CI-MPR, or CD-MPR antiserum.

 

Figure 4
View larger version (22K):
[in this window]
[in a new window]

  		Figure 4. Secretory release of [35S]sulfate-labeled serglycin is enhanced in the presence of PMA and decreased through the action of tunicamycin. U937 cells were labeled with [35S]sulfate for 10 min and chased for up to 1 h in the absence or presence of the indicated drugs. [35S]Sulfate-labeled serglycin was precipitated by TCA from cells and media and quantified by liquid scitillation counting. In this experiment, approximately one-third of [35S]sulfate-labeled serglycin was secreted from control cells within 1 h of chase. This amount was increased drastically through PMA treatment (0.2 µM), whereas treatment with tunicamycin (6 µg/ml) greatly enhanced lysosomal targeting of serglycin in the absence as well as in the presence of PMA. Pretreating cells with tunicamycin for 24 h decreased their ability to sulfate serglycin by ~50%, which was mathematically corrected to enable a convenient presentation.

 
We then used cross-linking with DSP to inquire whether the MPRs and sortilin bound to serglycin during the sorting process within the TGN. U937 cells were labeled with [35S]sulfate for 30 min and then cross-linked with DSP or mock-incubated. Aliquots of the cell extracts were subjected to immunoprecipitation, which were analyzed by SDS-PAGE and fluorography (Fig. 3B , right panel).

In another experiment, U937 cells were labeled with [35S]sulfate for 10 min to label Golgi-resident molecules followed by DSP cross-linking. Two aliquots of the cell extract were used to immunoprecipitate each of the following antigens: lysozyme, the CD-MPR, or the CI-MPR. As a control, two aliquots of the cell extract were treated with nonimmune serum, followed by SDS-PAGE analysis and fluorography (Fig. 3C) .

Without cross-linking, almost none of the applied antibodies was able to coimmunoprecipitate serglycin. Only the CI-MPR antiserum showed a trace of coimmunoprecipitated serglycin. Application of DSP resulted in strong cross-linking and coimmunoprecipitation of lysozyme and [35S]sulfate-labeled serglycin. This can be expected from its known affinity for serglycin [19 ]. Compared with lysozyme, the CI-MPR showed a weaker but distinct cross-linking to [35S]sulfate-labeled serglycin, whereas cross-linking of sortilin appeared much weaker than that of the CI-MPR, which is sulfated itself and thus, can be seen in Figure 3B and 3C , as labeled polypeptide. Cross-linking of lysozyme and the CI-MPR to [35S]sulfate-labeled serglycin was observed consistently in several other experiments, whereas appreciable cross-linking of the CD-MPR to [35S]sulfate-labeled serglycin was never observed (Fig. 3C) .

Tunicamycin greatly augments lysosomal transport of serglycin
As the CI-MPR binds primarily to M6P-bearing ligands with high affinity, we eliminated these ligands and examined its impact on CI-MPR-mediated lysosomal transport of serglycin. U937 cells were labeled with [35S]sulfate for 15 min followed by a chase period for up to 60 min in the absence or presence of PMA or tunicamycin, as indicated (Fig. 4 ). Preincubation with PMA and tunicamycin started 3 and 24 h prior to labeling, respectively.

[35S]Sulfate-labeled serglycin was precipitated from cell extracts and media with the help of TCA and quantified by liquid scintillation counting.

Part of the newly synthesized serglycin was secreted from control cells, whereas serglycin, remaining within the cells, was degraded within lysosomes (Fig. 4 , upper left panel and ref. [19 ]). Treatment of U937 cells with PMA resulted in almost complete secretion of serglycin (Fig. 4 , upper right panel). In the absence and presence of PMA, secretion of serglycin was diminished greatly after tunicamycin pretreatment (Fig. 4 , lower panels), indicating that lysosomal transport of serglycin was improved greatly.

Tunicamycin greatly enhanced cross-linking of serglycin to the CI-MPR
Next, we examined whether the CI-MPR was responsible for improved lysosomal transport of serglycin. Therefore, U937 cells were or were not pretreated with tunicamycin for 24 h, labeled with [35S]sulfate for 10 min, and cross-linked with DSP. Aliquots of cell extracts were subjected to precipitation with TCA to quantify sulfation of serglycin. Lysozyme, sortilin, and the CI-MPR were then immunoprecipitated from aliquots of the remaining cell extracts and TCA and immunoprecipitates were analyzed by liquid scintillation counting and by SDS-PAGE followed by fluorography (Fig. 5 ).


Figure 5
View larger version (28K):
[in this window]
[in a new window]

  		Figure 5. Tunicamycin greatly augments cross-linking of the CI-MPR to [35S]sulfate-labeled serglycin. U937 cells were incubated with (+Tm) or without (–Tm) tunicamycin for 24 h, labeled with [35S]sulfate for 10 min, and treated with cross-linker (DSP) for another 10 min. Lysozyme, sortilin, and the CI-MPR were then immunoprecipitated (IP) from the cell extracts and analyzed by reducing SDS-PAGE and fluorography. TCA-precipitated, [35S]sulfate-labeled serglycin from aliquots of the cell extracts was included in this analysis too. Tunicamycin treatment inhibited sulfation of serglycin, as revealed by TCA precipitation and immunoprecipitation of lysozyme. The relative amount of [35S]sulfate-labeled serglycin, immunoprecipitated with the CI-MPR, was increased threefold in the presence of tunicamycin.

 
Tunicamycin caused an overall 1.7-fold reduction of serglycin sulfation, as determined by TCA precipitation (see also Fig. 5 ). In contrast, the CI-MPR-precipitable serglycin signal was increased 1.8-fold by tunicamycin, resulting in a 3.1-fold increase of the relative serglycin signal cross-linked to the CI-MPR. This indicates that binding of serglycin to the CI-MPR is improved after depletion of M6P-bearing ligands. In turn, this may be responsible for the increased lysosomal delivery of serglycin as indicated in Figure 4 . It also shows that M6P-bearing ligands have a higher affinity toward the CI-MPR than serglycin and that depletion of these ligands opens up a new binding site to which serglycin can additionally bind, thus improving binding of serglycin to this receptor.

Sulfation of the CI-MPR was inhibited in the presence of tunicamycin, which may indicate that [35S]sulfate-labeled serglycin was cross-linked to previously synthesized CI-MPR molecules bearing unlabeled sulfate and that the CI-MPR synthesized in the presence of tunicamycin was not available for sulfation during the labeling period.

PMA affects sorting of M6P-bearing lysosomal enzymes
In this experiment, we examined whether PMA would also influence sorting of regular, M6P-bearing lysosomal enzymes. Therefore, U937 cells were labeled with a mix of L-[35S]methionine and L-[35S]cysteine overnight in the absence and presence of PMA. CD was then immunoprecipitated from the cell and medium extracts. The immunoprecipitates were analyzed by SDS-PAGE and visualized by fluorography (Fig. 6 ).


Figure 6
View larger version (58K):
[in this window]
[in a new window]

  		Figure 6. PMA increases secretion of cathepsin D. Two cultures of U937 cells were or were not treated with 0.2 µM PMA while being labeled with a mix of L-[35S]methionine and L-[35S]cysteine overnight. Cathepsin D was immunoprecipitated from cell and media extracts and visualized by SDS-PAGE followed by fluorography. The order of lanes was different on the original fluorogram and was rearranged for clearer presentation. The pro (pCD), intermediate (iCD), and mature (mCD) forms of CD are indicated in the margin as well as the position of molecular mass standards.

 
Upon PMA treatment, targeting of CD to lysosomes was diminished slightly, and secretion was increased moderately. Compared with serglycin, lysosomal targeting of CD was less affected by PMA, indicating that CI-MPR-mediated lysosomal transport was still going on but was performed with lower efficiency. Taken together, the results show that PMA moderately increased secretion of the high-affinity ligand CD (Fig. 6) but caused an almost complete secretion of the low-affinity ligand serglycin (Fig. 4) .

The formation of TGN-derived lysosomal transport vesicles is reduced by PMA
PMA is known to induce massive morphological changes in leukocytic cells, such as neutrophils [24 ] or U937 cells [25 ], within minutes of application. One of these changes is a greatly stimulated generation of multivesicular bodies (MVBs) in U937 cells [25 ]. Secretory release of lysozyme is also up-regulated to almost 100% within a few minutes of PMA application to U937 cells [26 ], indicating that transport of serglycin, which mediates lysosomal delivery of lysozyme [19 ], may be affected by these changes too, e.g., by altered vesicular trafficking of the CI-MPR. We therefore compared the distribution of newly synthesized serglycin, proCD, and lysozyme in sucrose density gradients after the following pulse/chase protocol.

U937 cells were preincubated with or without PMA, labeled with [35S]sulfate for 5 min, and chased for 10 more min in the continued absence or presence of PMA. As sulfation takes place in the trans-Golgi, secretory and lysosomal transport vesicles will be loaded with [35S]sulfate-labeled serglycin and detach from the TGN during the chase period. Cells were then opened by nitrogen cavitation, and the postnuclear supernatants were adjusted to 50% sucrose and layered beneath a sucrose gradient. Centrifugation of the gradient resulted in floating of intact vesicles into the gradient, whereas cytosolic components and the contents of ruptured vesicles remained at the bottom. Following fractionation, the content of [35S]sulfate-labeled serglycin was determined in each fraction by precipitation with TCA and liquid scintillation counting as shown in Figure 7A .


Figure 7
View larger version (34K):
[in this window]
[in a new window]

  		Figure 7. PMA reduces formation of lysosomal transport vesicles. (A) To characterize secretory and lysosomal transport vesicles detaching from the TGN, U937 cells were ({square}) or were not ({diamondsuit}) pretreated with PMA for 3 h, labeled with [35S]sulfate for 5 min, and chased for 10 min in the continued absence or presence of PMA. Cells were then opened by nitrogen cavitation, and postnuclear supernatants of the cells were layered under a linear, 18–40% sucrose gradient, centrifuged for 18 h at 200,000 g at 4°C, and fractionated. The content of [35S]sulfate-labeled serglycin in the fractions was determined by TCA precipitation and liquid scintillation counting. The numbering of fractions applies to all panels of the figure. (B and C) U937 cells were pretreated for 3 days with calcitriol, which increases expression of lysozyme and lysosomal enzymes without interfering with their lysosomal targeting. Cells were then labeled with a mix of L-[35S]methionine and L-[35S]cysteine for 1 h in the absence or presence of PMA. After a chase period of 15 min, cells were opened by nitrogen cavitation, and the postnuclear supernatants were subjected to the same sucrose density centrifugation procedure as described above. After fractionation, CD and lysozyme were immunoprecipitated simultaneously from the fractions and analyzed by SDS-PAGE. *, Immunoprecipitation contaminant and not the intermediate form of CD, as the latter should appear closer below proCD (see Fig. 6 ).

 
In control cells, two types of serglycin-containing vesicles were generated: a light type of vesicle (Fractions 1–4), containing approximately two-thirds of labeled serglycin, and a heavier type of vesicle (Fractions 4–6), containing approximately one-third of labeled serglycin. In the presence of PMA, the amount of serglycin in light fractions was reduced greatly, suggesting that they contained lysosomal transport vesicles, whereas the heavier fractions most likely contained secretory vesicles.

To obtain further insight into the contents of these fractions, U937 cells were pretreated for 3 days with calcitriol, which increases expression of lysozyme and lysosomal enzymes without interfering with their lysosomal targeting. Cells were then labeled with a mix of L-[35S]methionine and L-[35S]cysteine for 1 h in the absence or presence of PMA. After a chase period of 15 min in the continued absence or presence of PMA, cells were opened by nitrogen cavitation and subjected to the same sucrose density centrifugation protocol as described above. CD and lysozyme were then immunoprecipitated simultaneously from all the fractions and analyzed by SDS-PAGE (Fig. 7B and 7C) .

Treatment of U937 cells with PMA severely reduced the cell-associated amount of newly synthesized lysozyme, as expected, and caused a moderate decrease in the cellular content of newly synthesized proCD. Both enzymes were especially depleted in the upper part of the gradient (Fractions 1–3), suggesting again to contain lysosomal transport vesicles. Upon isolating clathrin-coated vesicles from untreated U937 cells using clathrin-specific antibodies, we detected proCD and lysozyme in these vesicles (not shown).

From these data, we conclude that the formation of CI-MPR containing lysosomal transport vesicles was diminished greatly in the presence of PMA, which is known to activate protein kinase C in U937 cells [26 ]. These observations are consistent with the known, clathrin-dependent diversion of MPRs and lysosomal enzymes from the TGN and suggest that lysozyme, serglycin, the CI-MPR, and proCD are delivered to lysosomes in the same lysosomal transport vesicles. This is underscored by the fact that lysozyme as well as the CI-MPR can be cross-linked to serglycin in these vesicles after labeling U937 cells with [35S]sulfate for 10 min followed by chase periods of 15 and 30 min (not shown).

The CI-MPR is cross-linked to serglycin of HL-60 cells
Here, we examined whether the CI-MPR may contribute to segregation of serglycin from the secretory pathway in the azurophilic granule generating human promyelocytic HL-60 cell line. One of the major granular constituents in these cells is MPO, which was shown to bind to serglycin while being delivered to azurophilic granules [4 ].

HL-60 cells were labeled with [35S]sulfate for 30 min, cross-linked with DSP for 10 min, and lysed. The cell lysate was subjected to immunoprecipitation with nonimmune, antilysozyme, anti-MPO, and anti-CI-MPR serum. Immunoprecipitates were analyzed by liquid scintillation counting and SDS-PAGE followed by fluorography (Fig. 8 ).


Figure 8
View larger version (47K):
[in this window]
[in a new window]

  		Figure 8. Cross-linking of serglycin in HL-60 cells, which were labeled with [35S]sulfate for 30 min, washed, and treated with 1 mM DSP for 10 min at 37°C. Aliquots of the cell lysate were subjected to immunoprecipitation with nonimmune serum (Co), lysozyme, CI-MPR, and MPO antiserum. The immunoprecipitable radioactivity was analyzed by liquid scintillation counting (one-third of the sample) and by reducing SDS-PAGE and fluorography (two-thirds of the sample).

 
As expected, MPO was cross-linked heavily with serglycin. Compared with MPO, the CI-MPR roughly bound 15% of [35S]sulfate-labeled serglycin, whereas lysozyme showed only little cross-linking to serglycin. This indicates that the CI-MPR may be involved in targeting of serglycin in different cellular settings, as azurophilic granule-forming HL-60 cells are distinct from the promonocytic U937 cell line, which primarily contains secretory lysosomes. It is interesting that cross-linking of the CI-MPR to serglycin was even better than that of lysozyme. This may be explained by lower expression levels of lysozyme in HL-60 cells. In addition, lysozyme has a lower affinity for CS than MPO [4 ]. For unknown reasons, sulfation of the CI-MPR was not detected in HL-60 cells under the conditions of this experiment.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In our study, we found that lysosomal delivery of serglycin in human promonocytic U937 cells was mediated by its CS moiety (Fig. 1) . From the ongoing lysosomal transport of CS in the absence of protein biosynthesis, we concluded that sorting of CS and probably of serglycin too is dependent on a recycling receptor system that is not easily depleted by cycloheximide treatment.

Therefore, we examined whether serglycin is delivered to lysosomes by known lysosomal-sorting receptors, i.e., by sortilin, the CI-MPR, the CD-MPR, or a combination thereof. 125I-Labeled sortilin and the 125I-labeled CI-MPR bound to CS-sepharose and were eluted by 150–250 mM NaCl and 100–450 mM NaCl, respectively (Fig. 2) , indicating that they may interact with the GAG portion of serglycin similar to other proteins, such as lysozyme or MPO [4 , 19 ].

Immunocytochemistry of U937 cells showed that the CI-MPR colocalized with serglycin to a large extent (Fig. 3A) . In addition, [35S]sulfate-labeled serglycin was coimmunoprecipitated with the CI-MPR and to a much lesser extent, with sortilin after labeling U937 cells with [35S]sulfate and cross-linking with DSP (Fig. 3B and 3C) . Lysosomal transport of serglycin could be modulated by tunicamycin and PMA (Fig. 4) , agents that greatly influence ligand binding and cellular trafficking of the CI-MPR, respectively. By inhibiting synthesis of M6P-bearing oligosaccharides of lysosomal enzymes in the presence of tunicamycin, binding of serglycin to the CI-MPR was enhanced approximately threefold (Fig. 5) and resulted in enhanced lysosomal transport of serglycin (Fig. 4) . Conversely, PMA decreased the generation of CI-MPR-containing lysosomal transport vesicles (Fig. 7) . This led to a massive mis-sorting of the low-affinity ligand serglycin (Fig. 4) and a moderate mis-sorting of the high-affinity ligand CD (Fig. 6) . Mis-sorting of CI-MPR ligands was not a result of redirection of the CI-MPR from the TGN to the plasma membrane, as inclusion of 5 mM M6P in the culture medium did not increase the amount of secretory proCD nor did PMA increase the amount of cell-surface iodinatable CI-MPR (not shown). It is interesting that treatment of cells with tunicamycin reversed almost entirely the PMA-induced secretion of serglycin (Fig. 4) . This suggests that increasing the affinity of serglycin for the CI-MPR can balance the reduced formation of lysosomal transport vesicles, thus bringing back lysosomal delivery of serglycin close to normal.

As shown by Nilsson and others [25 ], treatment of U937 cells with PMA induces massive morphological changes of U937 cells as a result of changes in vesicular traffic. The most prominent effect is a pronounced production of MVBs. Another change was the reduced production of lysosomal transport vesicles (Fig. 7) . It is interesting that Baldassarre and coworkers [27 ], who treated rat basophilic leukemia cells with PMA, observed complete mis-sorting, i.e., quantitative secretion of serglycin and proCD, indicating that in these cells, too, endosomal transport of these ligands may be linked to the CI-MPR and that endosomal transport of the CI-MPR was probably abolished completely.

The contribution of sortilin to lysosomal targeting of serglycin is probably minor, as its affinity toward CS and cross-linking to serglycin was quite weak as compared with the CI-MPR. The influence of the CD-MPR on sorting of serglycin is even less, as the CD-MPR did not cross-link to serglycin nor did it bind to CS-sepharose.

Here, we show that serglycin is a novel ligand for the CI-MPR in lysosomal transport. This finding extends the list of ligands of this unique receptor. Serglycin appears to act as an adaptor collecting positively charged, soluble proteins for CI-MPR-mediated lysosomal/granular transport. Our findings indicate that serglycin and M6P-bearing proteins bind to different domains of the CI-MPR. As binding of serglycin can be enhanced greatly by eliminating M6P-bearing proteins, we conclude that the latter have a higher affinity toward their binding domain than serglycin. After eliminating M6P-bearing proteins, serglycin can then bind to the unoccupied M6P-binding region, which increases the affinity of serglycin greatly toward the CI-MPR. It may be that the M6P-binding pocket, which is designed to bind tightly to phosphate, also has a considerable affinity for sulfate groups.

In an earlier study [19 ], it was shown that lysosomal transport of the serglycin-binding protein lysozyme was insensitive to NH4Cl, whereas secretion of the M6P-bearing lysosomal enzyme cathepsin D was enhanced greatly by NH4Cl. This indicates that detaching serglycin/enzyme complexes from the CI-MPR is not dependent on acidification of endosomes. As serglycin is a low-affinity ligand of the CI-MPR, detachment of serglycin from the receptor can probably be achieved by mere dilution. The following observation speaks in favor of this hypothesis: Although the CI-MPR is expressed on the plasma membrane of U937 cells and is able to take up M6P-bearing lysosomal enzymes by endocytosis, this could not be observed for [35S]sulfate-labeled serglycin (not shown). This suggests that binding of serglycin to the CI-MPR in the TGN requires relatively high concentrations of the CI-MPR and serglycin and perhaps other factors, which have not been determined in this study.

Targeting of serglycin to lysosomes or lysosome-like organelles through the CI-MPR is especially important in hematopoietic cells and explains the colocalization of classical lysosomal enzymes and positively charged microbicidal enzymes plus serglycin in these organelles [28 ]. In some cell types, particularly in maturing neutrophil granulocytes, serglycin is degraded after having delivered its accompanying proteins to azurophilic or other granules [29 ]. Thus, function and fate of serglycin in these cells resemble very much those in U937 cells. It seems that if further delivery tasks are needed, e.g., the delivery of granzymes and perforin to target cells of cytotoxic T lymphocytes, serglycin is stored within its respective organelle and is secreted as complex with its cytotoxic cargo [30 ].

It may be that additional transporters can be found for lysosomal/granular delivery of microbicidal polypeptides in the various hematopoietic cell types and that biogenesis of granules relies on several transport systems, which may complement or substitute each other. With the identification of the CI-MPR as a potential serglycin sorter, at least a first step was taken toward elucidating the molecular components of this cellular sorting machinery.


   ACKNOWLEDGEMENTS
 
We are indebted to the colleagues who provided the antibodies to the CI-MPR (Kurt von Figura), serglycin (Carsten U. Niemann), cathepsin D (Stephen Weitz), and sortilin (Claus M. Petersen). The technical assistance of Heinrich Kaiser (cell culture) and Thomas Stein (gel electrophoresis) is greatly acknowledged. Special thanks go to Thomas Schrader for his continuous support and encouragement.

Received August 17, 2006; revised November 2, 2006; accepted November 28, 2006.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Kolset, S. O., Prydz, K., Pejler, G. (2004) Intracellular proteoglycans Biochem. J. 379,217-227[CrossRef][Medline]
  2. Forsberg, E., Pejler, G., Ringvall, M., Lunderius, C., Tomasini-Johansson, B., Kusche-Gullberg, M., Eriksson, I., Ledin, J., Hellman, L., Kjellén, L. (1999) Abnormal mast cells in mice deficient in a heparin-synthesizing enzyme Nature 400,773-776[CrossRef][Medline]
  3. Åbrink, M., Grujic, M., Pejler, G. (2004) Serglycin is essential for maturation of mast cell secretory granule J. Biol. Chem. 279,40897-40905[Abstract/Free Full Text]
  4. Lemansky, P., Gerecitano-Schmidek, M., Das, R. C., Schmidt, B., Hasilik, A. (2003) Targeting myeloperoxidase to azurophilic granules in HL-60 cells J. Leukoc. Biol. 74,542-550[Abstract/Free Full Text]
  5. Grujic, M., Braga, T., Lukinius, A., Eloranta, M. L., Knight, S. D., Pejler, G., Abrink, M. (2005) Serglycin-deficient cytotoxic T lymphocytes display defective secretory granule maturation and granzyme B storage J. Biol. Chem. 280,33411-33418[Abstract/Free Full Text]
  6. Biederbick, A., Licht, A., Kleene, R. (2003) Serglycin proteoglycan is sorted into zymogen granules of rat pancreatic acinar cells Eur. J. Cell Biol. 82,19-29[CrossRef][Medline]
  7. Ghosh, P., Dahms, N. M., Kornfeld, S. (2003) Mannose 6-phosphate receptors: new twists in the tale Nat. Rev. Mol. Cell Biol. 4,202-212[CrossRef][Medline]
  8. Petersen, C. M., Nielsen, M. S., Nykjaer, A., Jacobsen, L., Tommerup, N., Rasmussen, H. H., Roigaard, H., Gliemann, J., Madsen, P., Moestrup, S. K. (1997) Molecular identification of a novel candidate sorting receptor purified from human brain by receptor-associated protein affinity chromatography J. Biol. Chem. 272,3599-3605[Abstract/Free Full Text]
  9. Nielsen, M. S., Madsen, P., Christensen, E. I., Nykjaer, A., Gliemann, J., Kasper, D., Pohlmann, R., Petersen, C. M. (2001) The sortilin cytoplasmic tail conveys Golgi-endosome transport and binds the VHS domain of the GGA2 sorting protein EMBO J. 20,2180-2190[CrossRef][Medline]
  10. Mazella, J., Zsurger, N., Navarro, V., Chabry, J., Kaghad, M., Caput, D., Ferrera, P., Vita, N., Gully, D., Maffrand, J. P., Vincent, J. P. (1998) The 100-kDa neurotensin receptor is gp95/sortilin, a non-G-protein-coupled receptor J. Biol. Chem. 273,26273-26276[Abstract/Free Full Text]
  11. Nielsen, M. S., Jacobsen, C., Olivecrona, G., Gliemann, J., Petersen, C. M. (1999) Sortilin/neurotensin receptor-3 binds and mediates degradation of lipoprotein lipase J. Biol. Chem. 274,8832-8836[Abstract/Free Full Text]
  12. Chen, Z. Y., Ieraci, A., Teng, H., Dall, H., Meng, C. X., Herrera, D. G., Nykjaer, A., Hempstead, B. L., Lee, F. S. (2005) Sortilin controls intracellular sorting of brain-derived neurotrophic factor to the regulated secretory pathway J. Neurosci. 25,6156-6166[Abstract/Free Full Text]
  13. Shi, J., Kandror, K. V. (2005) Sortilin is essential and sufficient for the formation of Glut4 storage vesicles in 3T3–L1 adipocytes Dev. Cell 9,99-108[CrossRef][Medline]
  14. Lefrancois, S., Zeng, J., Hassan, A. J., Canuel, M., Morales, C. R. (2003) The lysosomal trafficking of sphingolipid activator proteins (SAPs) is mediated by sortilin EMBO J. 22,6430-6437[CrossRef][Medline]
  15. Jacobsen, L., Madsen, P., Moestrup, S. K., Lund, A. H., Tommerup, N., Nykjaer, A., Sottrup-Jensen, L., Gliemann, J., Petersen, C. M. (1996) Molecular characterization of a novel hybrid receptor that binds the {alpha}2-macroglobulin receptor-associated protein J. Biol. Chem. 271,31379-31383[Abstract/Free Full Text]
  16. Hermey, G., Riedel, I. B., Hampe, W., Schaller, H. C., Hermans-Borgmeyer, I. (1999) Identification and characterization of SorCS, a third member of a novel receptor family Biochem. Biophys. Res. Commun. 266,347-351[CrossRef][Medline]
  17. Kikuno, R., Nagase, T., Ishikawa, K., Hirosawa, M., Miyajima, N., Tanaka, A., Kotani, H., Nomura, N., Ohara, O. (1999) Prediction of the coding sequence of unidentified genes. XIV. The complete sequences of 100 new cDNA clones from brain which code for large proteins in vitro DNA Res. 6,197-205[Abstract]
  18. Nagase, T., Kikuno, R., Ishikawa, K. I., Hirosawa, M., Ohara, O. (2000) Prediction of the coding sequences of unidentified human genes. XVI. The complete sequences of 150 new cDNA clones from brain which code for large proteins in vitro DNA Res. 7,65-73[Medline]
  19. Lemansky, P., Hasilik, A. (2001) Chondroitin sulfate is involved in lysosomal transport of lysozyme in U937 cells J. Cell Sci. 114,345-352[Abstract]
  20. Von Figura, K., Gieselmann, V., Hasilik, A. (1984) Antibody to mannose 6-phosphate specific receptor induces receptor deficiency in human fibroblasts EMBO J. 3,1281-1286[Medline]
  21. Laemmli, U. K. (1970) Cleavage of structural proteins during assembly of the head of bacteriophage T4 Nature 227,680-685[CrossRef][Medline]
  22. Laskey, R. A., Mills, A. D. (1975) Quantitative film detection of 3H and 14C in polyacrylamide gels by fluorography Eur. J. Biochem. 56,335-341[Medline]
  23. Imort, M., Zuhlsdorf, M., Feige, U., Hasilik, A., von Figura, K. (1983) Biosynthesis and transport of lysosomal enzymes in human monocytes and macrophages. Effects of ammonium chloride, zymosan and tunicvamycin Biochem. J. 214,671-678[Medline]
  24. Robinson, J. M., Badwey, J. A., Karnovsky, M. L., Karnovsky, M. J. (1987) Cell surface dynamics of neutrophils stimulated with phorbol esters or retinoids J. Cell Biol. 105,417-426[Abstract/Free Full Text]
  25. Nilsson, M., Nilsson, K., Forsbeck, K. (1989) Increased endocytosis and formation of multivesicular bodies in phorbol ester-stimulated human monoblastic U937 cells Exp. Cell Res. 181,551-565[CrossRef][Medline]
  26. Radons, J., Biewusch, U., Grässel, S., Geuze, H. J., Hasilik, A. (1994) Distinctive inhibition of the lysosomal targeting of lysozyme and cathepsin D by drugs affecting pH gradients and protein kinase C Biochem. J. 302,581-586
  27. Baldassarre, M., Dragonetti, A., Marra, P., Luini, A., Isidoro, C., Buccione, R. (2000) Regulation of protein sortine at the TGN by plasma membrane receptor activation J. Cell Sci. 113,741-748[Abstract]
  28. Burkhardt, J. K., Hester, S., Lapham, C. K., Argon, Y. (1990) The lytic granules of natural killer cells are dual-function organelles combining secretory and pre-lysosomal compartments J. Cell Biol. 111,2327-2340[Abstract/Free Full Text]
  29. Niemann, C. U., Cowland, J. B., Klausen, P., Askaa, J., Calafat, J., Borregaard, N. (2004) Localization of serglycin in human neutrophil granulocytes and their precursors J. Leukoc. Biol. 76,406-415[Abstract/Free Full Text]
  30. Veugelers, K., Motyka, B., Goping, I. S., Shostak, I., Sawchuk, T., Bleakley, R. C. (2006) Granule-mediated killing by granzyme B and perforin requires a mannose 6-phosphate receptor and is augmented by cell surface heparan sulfate Mol. Biol. Cell 17,623-633[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 GlycobiologyHome page
N. M Dahms, L. J Olson, and J.-J. P Kim
Strategies for carbohydrate recognition by the mannose 6-phosphate receptors
Glycobiology, September 1, 2008; 18(9): 664 - 678.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
jlb.0806520v1
81/4/1149    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Lemansky, P.
Right arrow Articles by Hasilik, A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Lemansky, P.
Right arrow Articles by Hasilik, A.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS