Identification of Mannose 6-Phosphate in Two Asparagine-linked Sugar Chains of Recombinant Transforming Growth Factor-/3l Precursor*

Recombinant transforming growth factor-81 (TGF- 81) precursor produced and secreted by a clone of Chinese hamster ovary cells was found to be glycosy- lated and phosphorylated. Treatment of 32P-labeled precursor protein with N-glycanase indicated that phosphate was incorporated into asparagine-linked complex carbohydrate moieties. Fractionation of 32P- labeled glycopeptides followed by amino acid sequence analysis indicated that greater than 95% of the label was incorporated into two out of three glycosylation sites at Asn-82 and Asn-136 of the TGF-81 precursor. Two-dimensional electrophoretic analysis of acid hydrolyzed precursor protein and precursor protein-de- rived glycopeptides indicated that 32P was incorporated as mannose 6-phosphate. Binding studies with the purified receptor for mannose 6-phosphate indicated that the TGF-01 precursor could bind to this receptor and the binding was specifically inhibited with mannose 6-phosphate. DNA sequence analysis of cDNA clones coding for monkey (I), human (2), and rodent (3) transforming growth factor-pl (TGF-Dl)’

DNA sequence analysis of cDNA clones coding for monkey (I), human ( 2 ) , and rodent (3) transforming growth factor-pl (TGF-Dl)' have indicated that this protein is synthesized as a large precursor, the carboxyl terminus of which is cleaved to yield the mature TGF-pl monomer. The biologically active TGF-P1 is a homodimer with a molecular weight of 24,000, consisting of two identical disulfide-linked chains of 112 amino acids each (4-6).
The amplified expression and secretion of mature TGF-01 and precursor proteins by Chinese hamster ovary cells has recently been reported (7). Analysis of [3H]glucosamine-and [32P]pho~phate-labeled serum-free supernatants from these cells demonstrated that TGF-B1 precursor proteins, but not mature TGF-01, were glycosylated and phosphorylated (8). Analysis of acid hydrolysates by thin layer electrophoresis showed that most of the phosphate was not bound to serine, threonine, or tyrosine residues.
In this report we show that all three potential asparaginelinked glycosylation sites  in simian TGF-Pl are used for carbohydrate addition and that phosphorylation occurs within the oligosaccharide side chains, suggesting a potential new function for the TGF-Dl precursor.

EXPERIMENTAL PROCEDURES AND RESULTS AND
DISCUSSION' Fig. 1A shows a line diagram illustrating the three forms of TGF-81 precursor proteins secreted by clone 17 cells, which have been previously described for the parental TGF-P-3-2000 cell line (7, 8).3 These proteins were characterized by site-specific anti-peptide antibodies as well as by direct protein sequencing. Also indicated in Fig. 1A are three potential asparagine-linked glycosylation sites predicted from the DNA sequence of the simian TGF-81 precursor located at amino acid residues 82, 136, and 176. Digestion of %3-labeled precursor proteins with N-glycanase resulted in a shift in migration of bands a and b to sharper and faster migrating bands, the largest of which had a molecular weight of approximately'39,OOO ( Fig. 2A, lune 2), consistent with the calculated molecular weight of 41,200 for the TGF-P1 precursor protein a. Digestion of 32P-labeled proteins with N-glycanase and subsequent fractionation of the digest by SDS-polyacrylamide gel electrophoresis indicated that the enzyme had removed all the label from the TGF-P1 precursor proteins (Fig. 2B, lune a ) , suggesting that 32P label was incorporated into asparagine-linked oligosaccharides.
The glycosylated and phosphorylated TGF-P1 precursor proteins a and b were subjected to cyanogen bromide cleavage and subsequent enzymatic digestion for further characteriza-Portions of this paper (including "Experimental Procedures," part of "Results and Discussion," Figs. S1-S3, and Table SI)  tion of the phosphorylation sites. The labeled glycopeptides were purified by gel permeation chromatography and reversed-phase high performance liquid chromatography. Sequence analysis of the three listed fragments indicated that Asn-82, Asn-136, and Asn-176 are glycosylated (Fig. 3). Over 95% of the label was found in peptides E(76-91) and E(134-139). Peptide T(174-180) contained less than 5% of the total incorporated 32P label.
Thin layer electrophoretic analysis of acid hydrolysates of total precursor proteins (a and b) as well as purified glycopep-A.  (Fig. 4, A-C). Comigration of peptideincorporated 32P label and standards of mannose 6-phosphate precursor proteins were hydrolyzed with acid for 2 h. Hydrolysis products were mixed with nonradioactive phosphoamino acids and mannose 6-phosphate and separated by electrophoresis at pH 1.9 and, orthogonally, at pH 3.5. An autoradiogram is shown. The sample is spotted at lower right (arrowhead). Man- Binding of 'a51-labeled TGF-@l precursor to the purified mannose 6-phosphate receptor. Purified human mannose 6-phosphate/IGF-II receptor (18) was adsorbed to microtiter wells coated with antibodies to this receptor. lZ6I-Labeled TGF-Dl precursor (100,000 cpm per well) was added in the presence of the indicated concentrations of unlabeled competitors. After 3 h at 4 "C, the wells were washed, cut out, and counted. Results are averages of duplicate wells that differed by less than 10% and are representative of three experiments. In wells containing no competitor (0% inhibition), 5.2% of the labeled ligand was bound. In wells not containing receptor, less than 0.12% of the labeled ligand was bound. TGF, TGF-61 precursor; MlP, mannose 1-phosphate; M6P, mannose 6-phosphate.
was observed upon electrophoresis in buffers at pH 1.9, pH 3.5, and pH 8.9 and in two different chromatography buffers ( Fig. 4 0 and data not shown). Acid hydrolysis may also generate mannose 6-phosphate from proteins modified with glycosyl-phosphatidylinositol (9). This has only been found at the carboxyl terminus of proteins and is therefore unlikely to account for mannose 6-phosphate in the TGF-Pl precursor.
We have shown that phosphorylation of the TGF-Pl precursor appears to be selective and to favor mannose in oligosaccharide chains at residues Asn-82 and Asn-136. Asn-176 is glycosylated, but only low level phosphorylation at mannose residues was observed.
The phosphorylated sugar analog, mannose 6-phosphate, appears to play a fundamental role in the targeted transport and intercellular exchange of lysosomal enzymes (reviewed in Ref. 10). Specific receptors that recognize the mannose 6phosphate residues of lysosomal enzymes have been identified and are essential components of the transport system. Lysosomal or related proteins that are secreted and contain mannose 6-phosphate have been identified in conditioned medium of tissue culture cells (11)(12)(13)(14). All of these proteins exhibit acid hydrolase activity. However, proliferin, a prolactin-related glycoprotein thought to be an autocrine growth regulator (15), was recently shown to bind to the mannose 6-phosphate receptor (16). This binding was specifically inhibited by mannose 6-phosphate (16). To determine whether the TGF-P1 precursor could also bind to the mannose 6-phosphate receptor, '261-labeled precursor was incubated with purified receptor. Approximately 30 times as much labeled ligand was bound to receptor as in control incubations without receptor. This binding was specific since it was 90% inhibited by either 100 nM TGF-pl precursor or 50 pM mannose 6-phosphate (Fig.  5 ) .