The Predominant Secreted Protein of Transformed Murine Fibroblasts Carries the Lysosomal Mannose 6-Phosphate Recognition Marker*

We have found that the major excreted protein (MEP) of transformed mouse fibroblasts, a phosphoglycoprotein of Mr = 35,000, carries the mannose 6-phosphate recognition marker. MEP secreted by Kirsten virus-transformed NIH 3T3 cells binds to a purified prepa- ration of lysosomal enzyme phosphomannosyl receptor, and this binding is specifically inhibited by mannose 6-phosphate. 32 pj introduced into MEP by meta-bolic labeling of intact cells is exclusively associated with asparagine-linked oligosaccharides as indicated by sensitivity to endohexosaminidase H. Labeling studies utilizing [2-3H]mannose indicate that approximately one-fifth of the mannose residues of MEP are phospho-rylated. Comparative studies of the synthesis, secretion, and uptake of MEP and of the lysosomal enzyme ,f-galactosidase indicate that MEP made by Kirsten virus-transformed NIH 3T3 cells is not handled in the same manner as are other lysosomal enzymes. MEP may be an unusual lysosomal protein, a mannose 6-phosphate-containing secretory protein, or both. Proteins secreted by transformed cultured fibroblasts differ significantly from those secreted by their nontransformed counterparts (1, 2). The secretion of some proteins such as the procollagens (3-5) is decreased, whereas the secretion of other proteins including plasminogen activator (6),

We have found that the major excreted protein (MEP) of transformed mouse fibroblasts, a phosphoglycoprotein of Mr = 35,000, carries the mannose 6-phosphate recognition marker. MEP secreted by Kirsten virustransformed NIH 3T3 cells binds to a purified preparation of lysosomal enzyme phosphomannosyl receptor, and this binding is specifically inhibited by mannose 6-phosphate. 32

pj introduced into MEP by metabolic labeling of intact cells is exclusively associated with asparagine-linked oligosaccharides as indicated by sensitivity to endohexosaminidase H. Labeling studies utilizing [2-3H]mannose indicate that approximately one-fifth of the mannose residues of MEP are phosphorylated. Comparative studies of the synthesis, secretion, and uptake of MEP and of the lysosomal enzyme ,f-galactosidase indicate that MEP made by Kirsten virus-transformed NIH 3T3 cells is not handled in the same manner as are other lysosomal enzymes. MEP may be an unusual lysosomal protein, a mannose 6phosphate-containing secretory protein, or both.
Proteins secreted by transformed cultured fibroblasts differ significantly from those secreted by their nontransformed counterparts (1,2). The secretion of some proteins such as the procollagens (3)(4)(5) is decreased, whereas the secretion of other proteins including plasminogen activator (6), a phosphoglycoprotein of Mr = 58,000 (2), and the major excreted protein of transformed mouse fibroblasts (1) is increased. In most cases, a role for these proteins in the establishment or maintenance of the transformed phenotype has not been defined.
We have been studying MEP,' a phosphoglycoprotein (7) whose secretion is increased 50-to 100-fold in many virally transformed mouse fibroblasts, including KNIH cells (1,8). A severalfold increase in synthesis and/or secretion of MEP has also been demonstrated with tumor promoters (8)(9)(10) and in mouse fibroblasts treated with platelet-derived growth factor (11), fibroblast growth factor (12), or epidermal growth factor (12). In mouse fibroblasts transformed virally or with tumor * 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. f Supported in part by an Arthritis Foundation Postdoctoral Fellowship.
¶ To whom requests for reprints should be sent. 'The abbreviations used are: MEP, major excreted protein of transformed murine fibroblasts; SDS, sodium dodecyl sulfate; PAGE, polyacrylamide gel electrophoresis; Eagle's medium, Eagle's minimal essential medium supplemented with antibiotics, nonessential amino acids, and 5% fetal bovine serum; endo H, endohexosaminidase H; Man-6-P, mannose 6-phosphate; KNIH cells, Kirsten virus-transformed NIH 3T3 cells; CHO, Chinese hamster ovary. promoters, it has been shown that increased secretion of MEP is accompanied by a concomitant increase in the level of translatable MEP mRNA (8).
Recently, Nilsen-Hamilton et al. (13) have shown that in nontransformed mouse 3T3 cells, secretion of MEP is stimulated by treatment of the cells with NH 4 CI and monensin. Since secretion of many secretory proteins is unaffected or decreased by these agents, whereas secretion of lysosomal enzymes is increased by them (14-18), we have been studying the relationship of MEP and lysosomal proteins.
The targeting of many lysosomal enzymes in cultured fibroblasts depends on the presence of mannose 6-P residues on these enzymes. These residues allow binding to the phosphomannosyl receptor, a membrane-associated glycoprotein (for reviews see Refs. [19][20][21]. We report here that MEP binds specifically to a purified preparation of the phosphomannosyl receptor, that phosphate metabolically incorporated into MEP is exclusively linked to endohexosaminidase H-sensitive oligosaccharides, and that acid hydrolysates of MEP contain mannose phosphate.

MATERIALS AND METHODS
Cell Culture-KNIH cells were obtained from C. Scher (Harvard Medical School) and maintained at 37 °C in 5% CO 2 in Dulbecco's modified Eagle's medium supplemented with penicillin (50 units/ml), streptomycin (50 g/ml), and 10% calf serum (Colorado Serum Co.). WTT2, a subclone of a proline auxotrophic CHO cell line, was obtained from A. Robbins (National Institutes of Health) and maintained at 34 C in 5% CO 2 in Eagle's minimal essential medium supplemented with antibiotics, nonessential amino acids, and 5% fetal bovine serum (GIBCO).
When KNIH cells were labeled with [4,5-3H]leucine, the following procedures were used. Cells are grown in 100-mm culture dishes to approximately 5 106 cells/dish. Before labeling, the cells were rinsed three times with Waymouth MAB 87-3 medium (22), as formulated in the GIBCO catalogue, with added antibiotics and 5% dialyzed fetal bovine serum, but with leucine reduced to 1.7 g/ml. Cells were labeled in 4 ml of the same medium containing 0.1 mCi/ml of [4,5-3H] leucine (Amersham Corp., 40 Ci/mmol). For pulse-chase experiments, labeling was terminated by the addition of 0.1 ml of L-leucine solution (5 mg/ml).
At the indicated times, the growth medium was collected and protein precipitated with 80% (NH 4 ) 2 S0 4 . The precipitate was dissolved in 1 ml of 10 mM sodium phosphate, pH 7.0, containing 0.15 M NaCI (Buffer A) and dialyzed for 16 h at 4 C against 1000 volumes of the same buffer. Man-6-P and Nonidet P-40 were added to the dialyzed samples to final concentrations of 10 mM and 1%, respectively. The cells were harvested by trypsinization, washed with Eagle's medium to remove trypsin, and extracted for 10 min at 0 C with 1 ml of Buffer A containing 10 m Man-6-P and 1% Nonidet P-40. The cell extracts and medium extracts were adjusted to a final volume of 2 ml and frozen.
Prior to immunoprecipitation of labeled proteins, the extracts were 11145 by guest on March 23, 2020

Uptake of MEP and ,8-Galactosidase by CHO cells-The
finding that MEP binds specifically to purified preparations of phosphomannosyl receptor prompted us to study the uptake of MEP by CHO cells. CHO cells have been used to study the uptake of lysosomal enzymes present in secretions of human skin fibroblasts and CHO mutants (34). Uptake of lysosomal enzymes by CHO cells is mediated exclusively by the phosphomannosyl receptor. When secretions from [ 3 H]leucine-labeled KNIH cells were added to CHO cells, fi-galactosidase, present in the secretions as the 84,000 form (Fig. 6, lane N), was efficiently taken up by the cells and processed to a 60,000 form (Fig. 7, lane G). f-Galactosidase present in NH 4 Cl-induced secretions of KNIH cells was also taken up by CHO cells, but to a lesser extent (lanes E and F). In contrast, MEP, in normal secretions or secretions from cells treated with NI-H 4 Cl, was not taken up as well by CHO cells (lanes A-D). The small amount of MEP found in the cells (lanes A and C, not clearly seen in this photograph) appeared as a lower molecular weight form of approximately 19,000-20,000 and represents 5-6% of the total labeled MEP recovered from the cells and medium.

DISCUSSION
The manyfold increase in MEP synthesis and secretion which correlates with transformation and tumor promotion in cultured mouse cells has allowed us to use this phenomenon as a molecular marker of the transformed phenotype (1,7,8). Until now, we have had no hint as to the possible function of this secreted phosphoglycoprotein. In this work, we show that MEP contains mannose phosphate residues and binds quantitatively and specifically to the phosphomannosyl receptor. This membrane-associated glycoprotein receptor has been implicated in the intracellular translocation of lysosomal enzymes (19)(20)(21).
The data presented here indicate that MEP has some similarities to other well studied lysosomal proteins. In previous studies, the mannose phosphate marker has been demonstrated to occur on lysosomal enzymes or glycoprotein fractions enriched in lysosomal enzymes. The recognition marker is thought to serve as a signal to direct these proteins to lysosomes. As for lysosomal enzymes, the mannose phosphate moieties of MEP occur on asparagine-linked oligosaccharides that can be released from the glycoprotein by treatment with endohexosaminidase H. These moieties are presumably synthesized in a manner similar to that for lysosomal enzymes, by transfer of GlcNAc-1-P from UDP-GlcNAc to the oligosaccharides to give phosphodiester moieties with the structure GlcNAc-P-Man and subsequent removal of terminal GlcNAc residues to give the phosphomonoester, P-Man (28-31). Most of the oligosaccharides of secreted MEP were sensitive to treatment with alkaline phosphatase, indicating that they contain mannose phosphate in the phosphomonoester form. A portion of the oligosaccharides also contained negatively charged moieties that were resistant to alkaline phosphatase. While not further characterized, such moieties may correspond to phosphodiester moieties found on lysosomal enzymes.
In contrast to the situation seen with a well characterized lysosomal protein, ,B-galactosidase (Fig. 6), in KNIH cells, MEP is almost quantitatively secreted into the culture medium. Thus, the presence of the phosphomannosyl recognition marker on MEP is apparently not sufficient for its efficient delivery and retention in lysosomes as is found for true lysosomal proteins. In addition, MEP is not taken up efficiently by CHO cells which are known to have surface phosphomannosyl receptors and which efficiently take up 8i-galactosidase synthesized by KNIH cells (Fig. 7). It is not clear why MEP binds to the phosphomannosyl receptor but is not efficiently internalized by CHO cells or targeted to lysosomes in KNIH cells, processes thought to be mediated by this same receptor. Perhaps MEP possesses the primary signal (Man-6-P residues) for binding to the receptor but is missing secondary structural features that are required for translocation of the receptor-bound ligand. Secondary structural features may include the number and/or orientation of Man-6-P residues on the oligosaccharides (35), other carbohydrate moieties on the oligosaccharides (27, 36), or structural determinants contained within the polypeptide portion of the glycoprotein (37). Alternatively, MEP may carry another signal specifying secretion which is not found on usual lysosomal proteins. Perhaps, the differently charged species of MEP which have been described (7) may each have different structural features which affect their subcellular distribution.
We have recently obtained evidence that in nontransformed mouse fibroblasts the portion of MEP retained within the cell is greater than in transformed cells and that this MEP has a predominantly lysosomal localization as determined by indirect immunofluorescence and electron microscopic localization. 2 Whether MEP is a novel mannose phosphate-containing secretory protein or a lysosomal protein, the localization of which has been altered by transformation, or both, is currently under investigation.