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J. Biol. Chem., Vol. 280, Issue 39, 33318-33323, September 30, 2005

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Identification of the Minimal Lysosomal Enzyme Recognition Domain in Cathepsin D^*

From the Department of Internal Medicine, Washington University School of Medicine, Saint Louis, Missouri 63110

Received for publication, June 1, 2005 , and in revised form, July 19, 2005.

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Specific recognition of lysosomal hydrolases by UDP-GlcNAc:lysosomal enzyme N-acetylglucosamine-1-phosphotransferase, the initial enzyme in the biosynthesis of mannose 6-phosphate residues, is governed by a common protein determinant. Previously, we generated a lysosomal enzyme recognition domain in the secretory protein glycopepsinogen by substituting in two regions (lysine 203 and amino acids 265–293 of the loop) from cathepsin D, a highly related lysosomal protease. Here we show that substitution of just two lysines (Lys-203 and Lys-267) stimulates mannose phosphorylation 116-fold. Substitution of additional residues in the loop, particularly lysines, increased phosphorylation 4-fold further, approaching the level obtained with intact cathepsin D. All the phosphorylation occurred at the carboxyl lobe glycan, indicating that additional elements are required for phosphorylation of the amino lobe glycan. These data support the proposal that as few as two lysines in the correct orientation to each other and to the glycan can serve as the minimal elements of the lysosomal enzyme recognition domain. However, our findings show that the spacing between lysines is flexible and other residues contribute to the recognition marker.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
The enzyme UDP-GlcNAc:lysosomal enzyme N-acetylglucosamine-1-phosphotransferase (abbreviated "phosphotransferase")³ has a central role in the targeting of newly synthesized lysosomal hydrolases to lysosomes. This enzyme recognizes a conformation-dependent protein determinant shared by the many different lysosomal hydrolases and then transfers N-acetylglucosamine 1-phosphate from UDP-GlcNAc to selected mannose residues of the high mannose oligosaccharides of the hydrolases. The N-acetylglucosamine residues are subsequently excised by "uncovering enzyme" generating mannose 6-phosphate monoesters that allow high affinity binding to mannose 6-phosphate receptors in the Golgi and translocation to lysosomes (1).

The nature of the common protein recognition determinant has received considerable attention because of its role in this targeting pathway. Our laboratory undertook a molecular dissection of the phosphotransferase recognition marker using a pair of aspartyl proteases, the lysosomal hydrolase cathepsin D and the secretory protein pepsinogen (2, 3). These proteins are 45% identical in amino acid sequence and share similar secondary and tertiary structure but differ in that cathepsin D is highly phosphorylated by phosphotransferase, whereas glycopepsinogen, the glycosylated form of pepsinogen, is not. By analyzing numerous chimeric proteins, we were able to define a phosphotransferase recognition patch in the carboxyl lobe of cathepsin D formed by two noncontinuous primary sequences, specifically Lys-203 and amino acids 265–293 that form a loop structure directed toward Lys-203 (2). Lysine residues 267 and 293 at the base of the loop made significant contributions to the degree of phosphorylation, implicating lysines as important elements to the recognition patch. It was further observed that the presence of additional regions of cathepsin D enhanced phosphorylation of the chimeric proteins, indicating that the recognition domain is a surface patch that contains multiple interacting sites and perhaps even a second independent site (4–6).

The critical role of lysines in defining the recognition domain has also been supported by studies from the Sahagian laboratory that have demonstrated a significant decrease in the mannose phosphorylation upon mutation of specific pairs of lysine residues in cathepsin D (Lys-203 and Lys-293) and cathepsin L (Lys-54 and Lys-99). Due to the fact that these lysine pairs are spaced 34 Å apart and positioned in a specific orientation relative to the target oligosaccharide in these proteases, they proposed a simple model in which just two critical lysine residues are necessary and sufficient for binding to phosphotransferase (7). However, because residual phosphorylation was observed in the absence of these critical lysine pairs, they pointed out that additional protein elements might be important. Further evidence for the role of lysines as components of the phosphotransferase recognition domain has come from mutagenesis studies of DNase I (8), aspartylglucosaminidase (9), and arylsulfatase (10).

In the current study, we have utilized both a gain-of-function (substituting cathepsin D residues into glycopepsinogen) and a loss-of-function (mutation of cathepsin D residues to alanine) approach in an attempt to determine the minimal phosphotransferase recognition domain. In contrast to our previous work that mostly utilized relatively large segments of sequence, we substituted single residues or small blocks of residues into glycopepsinogen to better analyze the role of individual amino acids in facilitating phosphorylation. Our experiments provide new insight into the nature of the phosphotransferase recognition marker.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Cell Lines, Plasmids, and Reagents—COS-1 and HeLa cells were obtained from ATCC and maintained in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum and 100 µg/ml penicillin and streptomycin. LoVo cells were also obtained from ATCC and maintained in Ham's F-12 medium containing 10% fetal bovine serum and 100 µg/ml penicillin and 100 units/ml streptomycin. Human glycopepsinogen cDNA and cathepsin D (bearing a C-terminal myc tag) cDNA were inserted into the pcDNA 3.1(+) expression vector. All mutations in these constructs were introduced using QuikChange site-directed mutagenesis (Stratagene) protocols. In the cathepsin D-myc experiments, lysine residues were always changed to alanine residues. Anti-myc polyclonal antibody (A-14) was from Santa Cruz Biotechnology. Anti-human pepsinogen antiserum was prepared as described previously (11). [2-³H]Mannose was purchased from PerkinElmer Life Sciences and [³⁵S]methionine/cysteine from MP Biomedicals. Concanavalin A-Sepharose was obtained from Amersham Biosciences, Protein A-agarose from Repligen Corp., and QAE-Sephadex from Sigma. Recombinant endoglycosidase H fused to maltose-binding protein was from New England Biolabs. All other reagents were of the highest quality available and were purchased from Sigma or Fisher.

Transfection and Labeling of Cells—Cells cultured in 60-mm plates for 24 h (80–90% confluent) were transfected with 2–3 µg of DNA using Lipofectamine Plus reagent system (Invitrogen) in the absence of antibiotics. At 24 h post-transfection, cell monolayers were washed and labeled with 200 µCi of [2-³H]mannose in Dulbecco's modified Eagle's medium, 10% dialyzed fetal bovine serum, and 1 mM glucose at 37 °C for 2 h. Following addition of glucose and mannose up to 5 mM to stop mannose uptake, the cells were incubated for 4 h before the media was collected and cleared by centrifugation to remove cell debris. Ammonium chloride (10 mM) was routinely present during the labeling and chase periods, because it resulted in higher secretion of most of the mutant constructs.

Immunoprecipitation and Oligosaccharide Analysis—Cell media samples were immunoprecipitated, and oligosaccharides were isolated and analyzed essentially as described in detail previously (6). For the cathepsin D-myc experiments, anti-myc antibody (50 µl) was bound to Protein A-agarose beads prior to immunoprecipitation of labeled cathepsin D from the media. Oligosaccharides released by Endo H digestion of immunoprecipitated protein, which include the phosphorylated molecules, were treated with mild acid to remove any N-acetylglucosamine residues still attached to the phosphate moieties. The percent phosphorylation was calculated as cpm recovered in Endo H-released oligosaccharides with one or two phosphates/total cpm in Endo H-released oligosaccharides (phosphorylated plus neutral) plus 2(Endo H-resistant complex oligosaccharides). The values for the complex oligosaccharides were multiplied by 2 to correct for the fact that they contain 3 mannose residues versus an average of 6 mannose residues per high mannose oligosaccharide. In all cases, values for each oligosaccharide pool obtained in routine mock transfection experiments were subtracted prior to percent phosphorylation calculations for the constructs. The mock values for phosphorylated species were 5 cpm or less in the LoVo cell experiments. The analysis of Endo H-released oligosaccharides by QAE-Sephadex chromatography allows for the separation of oligosaccharides bearing one or two mannose 6-phosphate residues.

Acid-activated Autoactivation of Cathepsin D Constructs—HeLa cells transfected with the various cathepsin D-myc constructs were labeled with 0.5 mCi of [³⁵S]methionine/cysteine for 2 h and chased for 4 h by the addition of cold methionine (up to 10 mM). Secreted cathepsin D molecules were immunoprecipitated with anti-myc polyclonal antibody. Washed beads were resuspended in 50 mM Tris-glycine buffer, pH 3.5, in the absence or presence of 500 µM pepstatin A, and incubated for 20 min. at 37 °C. Correctly folded molecules undergo autoactivation with cleavage of their propieces at this pH. This process is blocked by the presence of the aspartyl protease inhibitor, pepstatin A. Following neutralization with 10–12 µl of 1.5 M Tris-HCl, pH 8.8, labeled molecules were resolved by 8% SDS-polyacrylamide gel electrophoresis and visualized by autoradiography.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Influence of Cell Type on Glycopepsinogen Phosphorylation—We first expressed glycopepsinogen and two constructs in COS, HeLa, and LoVo cells to determine which is the most appropriate to use in the study. LoVo cells lack "uncovering enzyme" activity and are unable to form mannose 6-phosphate monoesters required for binding to mannose 6-phosphate receptors (12). To obtain accurate values of mannose 6-phosphate formation, the transfected cells were labeled with [2-³H]mannose followed by immunoprecipitation of the secreted glycopepsinogen molecules. The [³H]mannose-labeled oligosaccharides were then analyzed for the level of phosphorylation as described previously (6).

The phosphorylation of secreted glycopepsinogen and two constructs (E203K/A267K/Q293K or "3K"; termed construct 5 and "3K + loop segments ABC"; termed construct 8) expressed in COS, HeLa, and LoVo cells is shown in TABLE ONE. All three cell types phosphorylated the two constructs much better than glycopepsinogen, but the HeLa and LoVo cells exhibited greater phosphorylation compared with COS cells. The LoVo cells produced the highest level of phosphorylation as well as the highest ratio of oligosaccharides with two mannose 6-phosphate residues versus one mannose 6-phosphate residue. Based on these preliminary results, we utilized LoVo cells for the subsequent studies of modified glycopepsinogen.

We have now tested a new series of constructs with the goal of identifying the minimal number of residues required to generate an effective phosphotransferase recognition motif (TABLE TWO). The focus was on Lys-203 and the components of the loop, residues 265–293. Fig. 1 is a schematic representation of cathepsin D showing the location of its two N-linked glycans, Lys-203, and the region 265–293. A portion of the loop has been divided into three segments, and the amino acid sequence of cathepsin D and pepsinogen in this region is shown.

View larger version (11K): [in this window] [in a new window]   FIGURE 1. Structural organization of cathepsin D and alignment of loop amino acid sequences of human cathepsin D and pepsinogen. The number of residues is designated with respect to cathepsin D. N-Linked glycosylation sites are indicated by arrows. Identical amino acids between cathepsin D (CD) and pepsinogen (GP) are boxed. The position of the five lysine residues present in the loop structure are noted along with the position of lysine 203 (asterisk). Note that pepsinogen lacks all the lysines.

Construct 5, containing lysines at positions 203, 267, and 293, was phosphorylated 3.4%, a 156-fold stimulation over glycopepsinogen. The cathepsin D -loop contains three additional lysines at its tip (positions 277, 281, and 284). Construct 6 was prepared to contain all six lysines (203, 267, 277, 281, 284, and 293). Its oligosaccharides were phosphorylated 5.4%, a 245-fold increment over the baseline and 12.8% of that obtained with cathepsin D. Constructs 7 and 8 contain lysines 203, 267, and 293 as well as -loop segments AB and ABC, respectively. When expressed in LoVo cells, the resultant proteins were phosphorylated 7.7% and 9.7%. It is of note that construct 8 is phosphorylated almost twice as well as construct 6 even though both contain the six lysine residues at the identical positions. This indicates that residues other than lysines contribute to the potency of the recognition determinant.

The two N-linked glycans of glycopepsinogen are located on different lobes of this bilobed molecule: glycan 70 resides on the amino lobe, whereas glycan 199 is on the carboxyl lobe. Previously we reported that Lys-203 plus the -loop elements preferentially stimulated phosphorylation of the carboxyl lobe oligosaccharide at position 199, although the amino lobe glycan was also phosphorylated (11). To determine if this was the case with the minimal recognition elements, constructs were prepared lacking either the amino- or carboxyl-lobe glycan. Constructs that lacked the amino lobe glycosylation signal at position 70 and contained Lys-203, Lys-267, and Lys-293 (construct 9) or these lysines plus -loop segments ABC (construct 10) were phosphorylated 9.2% and 21.7%, respectively, a 418-fold and 986-fold increment over glycopepsinogen (Fig. 2). Furthermore, the glycan on construct 10 contained mostly high mannose species with two mannose 6-phosphate residues, indicative of a strong phosphotransferase recognition signal. The carboxyl lobe glycan of cathepsin D is known to acquire two mannose 6-phosphate residues, whereas the amino lobe glycan mostly has species with one mannose 6-phosphate residue (5). Constructs 11 and 12, which have the same amino acid changes as constructs 5 and 8, respectively, but contain only the amino lobe glycan were undetectably phosphorylated. These findings show that virtually all the phosphorylation of these minimal constructs occurs on the carboxyl lobe glycan.

Effect of Replacing Lysine Residues in Cathepsin D on Phosphorylation—We previously reported that substitution of Lys-203 in cathepsin D with an alanine residue decreased phosphorylation by 15% (4). However, this determination used a Xenopus oocyte expression system and binding to a CI-MPR affinity column as a measure of mannose phosphorylation. This assay could underestimate the effect, because phosphorylation of only one of the two oligosaccharides is required for binding to the affinity column. Cuozzo et al. (7) found that a K203A substitution in cathepsin D resulted in a 42% decrease in phosphorylation using a COS cell expression assay. These authors also reported that a K293A substitution decreased phosphorylation by 13%, whereas K267A had no effect. The double mutant K203A/K293A produced a 69% decrease in phosphorylation.

View larger version (8K): [in this window] [in a new window]   FIGURE 2. Effect of minimal recognition elements on individual glycopepsinogen oligosaccharides. Constructs containing the N-linked glycosylation consensus sequence for the amino lobe oligosaccharide alone (position 70) or carboxyl lobe oligosaccharide alone (position 199) were generated and tested. Constructs 5, 9, and 11 have lysines at positions 203, 267, and 293, whereas constructs 8, 10, and 12 contain these lysines plus -loop segments ABC. The constructs differ in their complement of glycans as indicated in the figure (oligosaccharide is abbreviated "CHO"). The values are the average of two to four determinations. Note that all the mannose phosphorylation in these constructs occurred on the carboxyl lobe glycan.

View larger version (10K): [in this window] [in a new window]   FIGURE 3. Autoactivation of cathepsin D constructs. Transfected HeLa cells were metabolically labeled with [35S]methionine/cysteine as described under "Materials and Methods." Secreted cathepsin D molecules were immunoprecipitated, and the beads were resuspended in buffer at pH 3.5 in the absence or presence of pepstatin. At pH 3.5, properly folded cathepsin D undergoes autoactivation with cleavage of the propeptide. The resulting mature species migrates faster in the gel. The autoactivation is blocked by pepstatin. Following resolution by SDS-PAGE, bands were visualized by autoradiography. WtCD, wild-type cathepsin D-myc; 3KA, K203A, K267A, K293A; 4KA, 3KA plus K277A; 6KA, 4KA plus K281A, K284A. Note the loss of autoactivation in the 6KA construct.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

View this table: [in this window] [in a new window]   TABLE FOUR Distance between -carbons atoms of key lysine and asparagine residues in various lysosomal enzymes The distances between -carbons atoms were measured using Swiss-Pdb viewer.

Sahagian and co-workers (7, 14) have extensively analyzed the role of lysine residues in the phosphorylation of cathepsin D and cathepsin L. In the experiments with cathepsin D, substitution of Lys-203 and Lys-293 with alanines decreased phosphorylation by 42 and 13%, respectively, whereas substitution of Lys-267 with alanine had no effect. When Lys-203 and Lys-293 were both replaced with alanines, phosphorylation decreased to 31% of wild-type cathepsin D (7). Substitution of the other lysines of cathepsin D, either alone or in combination with the Lys-203/Lys-293 modification had either no effect or a small inhibitory effect. They also reported that Lys-54 and Lys-99 of the propiece of cathepsin L were required for efficient phosphorylation of this cysteine protease (14). By examining the crystal structures of cathepsin D and procathepsin L, the Sahagian group observed that the distance between the critical lysine residues in both molecules was 34 ± 0.5 Å (7). They also noted that, in both instances, one of the critical lysines was much closer to the N-linked glycan than the other, although Lys-203 of cathepsin D is only 9.71 Å from Asn-199, whereas Lys-54 of procathepsin L is 25.08 Å from Asn-221. Based on these findings, these investigators proposed a model for the phosphorylation signal consisting of two lysine residues, exposed on the surface of the protein, which are spaced apart by 34 Å and positioned in a specific orientation relative to the target oligosaccharide (7). Because N-linked oligosaccharides are flexible, they would not have to be located a precise distance from the critical lysines in order for their mannose residues to interact with phosphotransferase (15). They further suggested that the microenvironment of the critical lysines may influence the signals and noted that there appeared to be additional phosphorylation signals as well in view of the fact that removal of the two critical lysines from both procathepsin L and cathepsin D failed to completely abolish mannose phosphorylation.

Our findings support the proposal of Sahagian and colleagues that as few as two lysines in the correct orientation to each other and the carbohydrate chain can serve as a minimal phosphotransferase recognition site. However, our data with the various combinations of Lys-203, Lys-267, and Lys-293 are not consistent with the requirement that the lysines be separated by 34 Å (see TABLE FOUR). The best pair (Lys-203/Lys-267) is separated by 40 Å and Lys-267/Lys-293, which are separated by only 12.2 Å, is as stimulatory as Lys-203/Lys-293, which are spaced 34 Å apart. Furthermore, as discussed above, the presence of additional lysines and other amino acids of the -loop region enhanced phosphorylation 4-fold over that obtained with Lys-203/Lys-267 alone. This construct (construct 10) phosphorylated the oligosaccharide at Asn-199 half as well as occurs in cathepsin D. These data are in agreement with the previous studies indicating that lysine residues are critical elements of a more extensive recognition marker (4).

The results from studies of three other phosphotransferase substrates address this issue. The phosphorylation of the two Asn-linked oligosaccharides of bovine DNase I requires four lysine residues, Lys-27, Lys-50, Lys-74, and Lys-124. Importantly, Lys-27 selectively facilitates phosphorylation of the Asn-18 glycan, whereas Lys-74 only stimulates phosphorylation of the Asn-106 glycan, indicating that these two lysine residues are components of separate signals (8). Lys-50 also acts on the Asn-106 glycan and it is 27.1 Å from Lys-74, again illustrating that the spacing between lysines that stimulate phosphorylation of the same glycan is not fixed at 34 Å (16). The efficient phosphorylation of the glycans of aspartylglucosaminidase depends mostly on two lysines (Lys-183 and Lys-214) that are separated by 35 Å, with Lys-177 and Tyr-178 having a lesser role (9). Human arylsulfatase has eight lysine residues, only one of which (Lys-457) has been implicated in the interaction with phosphotransferase (10). The largest distance between Lys-457 and the other lysines is 25Å, establishing that there cannot be a fixed interlysine spacing of 34 Å in this lysosomal enzyme. Because mutation of Lys-457 to alanine only decreased mannose phosphorylation by 70%, there must be other elements in the recognition marker that contribute to efficient phosphorylation.

Taken together, the common feature of all these studies is that selected lysine residues of the target lysosomal enzymes play a critical role in the interaction with phosphotransferase. In all the cases, with the possible exception of arylsulfatase A, two or more lysines serve as critical elements of the binding site. However, the spacing between these lysines is not fixed, nor is the distance of the lysines to the Asn-linked glycan that is phosphorylated. In each instance, mutation of the critical lysines fails to completely abolish mannose phosphorylation, indicating that additional elements play a role in the interaction with phosphotransferase.

Although our study provides important clues into the nature of the recognition domain, it also raises an intriguing question. The finding that just two lysines can govern phosphotransferase binding might predict that some secreted glycoproteins with abundant lysine residues would be good substrates for phosphotransferase. In fact, a survey of three secreted glycoproteins with solved crystal structures (transferrin, ribonuclease B, and heparin cofactor II) reveals multiple surface lysine pairs whose spacing and orientation toward the oligosaccharides are nearly identical to that found in cathepsin D. Why are these secreted glycoproteins not acted upon by phosphotransferase? One can envision several mechanisms to account for this paradox. First, the secreted glycoproteins may contain elements that mask existing phosphotransferase recognition elements (for example, neighboring acidic residues capable of neutralizing key lysines). The existence of inhibitory residues in the vicinity of key lysines has been observed in murine DNase I (16). Second, secreted glycoproteins may have evolved "decoy" lysine pairs that allow binding to phosphotransferase in an orientation that positions their oligosaccharides such that they can't be efficiently phosphorylated. Third, the secretory glycoproteins may lack non-lysine elements that are required for phosphorylation. In this regard, it should be noted that, because glycopepsinogen and cathepsin D are 45% identical in their amino acid sequence, it is possible that they share non-lysine residues that facilitate phosphorylation. Finally, it may be that many secreted glycoproteins do undergo low levels of mannose phosphorylation that diverts a small percentage of the molecules to lysosomes where they are degraded. Special measures would have to be taken to detect this process. Experiments designed to test some of these possibilities are in progress and should shed further light of the elusive nature of this protein-protein interaction.

	FOOTNOTES

 
^* This work was supported by National Institutes of Health Grant CA08759 (to S. K.) and National Institutes of Health Training Grant 5T32-HL07088–30 (to R. S.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ Both authors contributed equally to this work.

² To whom correspondence should be addressed. Tel.: 314-362-8803; Fax: 314-362-8826; E-mail: skornfel{at}im.wustl.edu.

³ The abbreviations used are: phosphotransferase, UDP-GlcNAc:lysosomal enzyme N-acetylglucosamine-1-phosphotransferase; Endo H, endonuclease H.

	ACKNOWLEDGMENTS

 
We thank Walter Gregory for production of the anti-pepsinogen antiserum, Dr. Thomas Baranski for critical reading of the manuscript, and Dr. Jay Ponder for the determination of distances between the -carbon atoms of various residues.

	REFERENCES

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 

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This Article Abstract Full Text (PDF) All Versions of this Article: 280/39/33318    most recent M505994200v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Steet, R. Articles by Kornfeld, S. Search for Related Content PubMed PubMed Citation Articles by Steet, R. Articles by Kornfeld, S. Social Bookmarking                      What's this?