Identification of Amino Acids That Modulate Mannose Phosphorylation of Mouse DNase I, a Secretory Glycoprotein

Abstract

We have reported that bovine DNase I, a secretory glycoprotein, acquires mannose 6-phosphate residues on 12.6% of its Asn-linked oligosaccharides when expressed in COS-1 cells and that the extent of phosphorylation increases to 79.2% when lysines are placed at positions 27 and 74 of the mature protein (Nishikawa, A., Gregory, W., Frenz, J., Cacia, J., and Kornfeld, S. (1997) J. Biol. Chem. 272, 19408 -19412 106 on the surface of DNase I, indicating that residues present over a broad area influence the interaction with UDP-GlcNAc:lysosomal enzyme N-acetylglucosamine-1-phosphotransferase, which is responsible for the formation of mannose 6-phosphate residues on lysosomal enzymes.In a previous report, we demonstrated that bovine DNase I, a secretory glycoprotein of the pancreas and the salivary gland, acquires mannose 6-phosphate moieties on its Asn-linked oligosaccharides (1), confirming the findings of Cacia et al. (2) with human DNase I. However, the level of oligosaccharide phosphorylation (12.6%) was considerably less than that observed with lysosomal hydrolases (usually 50% or greater). This indicated that DNase I is a weak substrate for UDPGlcNAc:lysosomal enzyme N-acetylglucosamine-1-phosphotransferase (phosphotransferase), 1 the enzyme that synthesizes the Man-6-P recognition marker for targeting to lysosomes. Phosphotransferase is known to recognize a conformation-dependent protein determinant that is present in lysosomal hydrolases, but absent in most secretory glycoproteins (3). This determinant has been shown to involve a broad surface patch that includes critical lysine residues (4 -11). The binding of lysosomal hydrolases to phosphotransferase is followed by the transfer of GlcNAc-P from UDP-GlcNAc to selected mannose residues on the Asn-linked high mannose oligosaccharides of the hydrolases. The N-acetylglucosamine is then removed by N-acetylglucosamine-1-phosphodiester-␣-Nacetylglucosaminidase to generate the phosphomannosyl recognition determinant that allows binding to the mannose 6-phosphate receptors in the Golgi and subsequent targeting to lysosomes (12). The basal level of phosphorylation of bovine DNase I was shown to be dependent on three residues (Lys 50 , Lys 124 , and Arg 27 ), consistent with the finding that lysine residues are important components of the phosphotransferase recognition determinant on lysosomal hydrolases (1). When Arg 27 was replaced with a lysine, oligosaccharide phosphorylation increased to 54%, demonstrating that phosphotransferase prefers lysine residues over arginines. Furthermore, mutation of Asn 74 to a lysine also increased phosphorylation to 50.3%, and the double mutant (R27K/N74K) was phosphorylated 79.2%, equivalent to the values observed with authentic lysosomal hydrolases. Interestingly, the lysine at position 27 specifically stimulated phosphorylation of the oligosaccharide at Asn 18 , whereas the lysine at position 74 selectively stimulated phosphorylation of the oligosaccharide at Asn 106 . The R27K and N74K bovine constructs were initially prepared bec...

A Necessary and Sufficient Determinant for Protein-selective Glycosylation in Vivo

“…Like GLuc-Trf-CA6 (11)(12)(13)(14)(15)(16)(17)(18)(19), GLuc-Trf-CA6(1-10) is modified with GalNAc when expressed in ␤GT3/CHO and ␤GT4/CHO cells (Fig. 6, A and B) but not to the same extent as GLuc-Trf-CA6 (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19). Furthermore a construct containing the carboxylterminal 14 amino acids of CA6, GLuc-Trf-CA6(6 -19) was also examined (Fig.…”

“…5, A and B). Adding the carboxyl-terminal 19 amino acids from CA6 to GLuc-Trf yields a form of Trf, GLuc-Trf-CA6 (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19) that is modified to a greater extent than GLuc-␣ with GalNAc by both ␤GT3 and Chimeric glycoproteins consisting of amino-terminal GLuc followed by ␣ or Trf and the epitope tag MycHis at the carboxyl terminus (see Fig. 1) were prepared and expressed in either ␤GT3/CHO (A) or ␤GT4/CHO (B) cells.…”

“…The addition of unique carbohydrate structures to select glycoproteins requires a "recognition determinant," a feature encoded in the peptide portion of these glycoproteins, to be recognized by one or more of the transferases responsible for their synthesis. In some cases key features of the peptide for such recognition determinants have been identified (7)(8)(9)(10)(11). However, understanding the molecular basis for recognition ultimately requires that a recognition determinant that does not itself comprise the site of modification can be added to a structurally unrelated glycoprotein and confer selective modification of this unrelated glycoprotein.…”

Role of N-Linked Oligosaccharide Flexibility in Mannose Phosphorylation of Lysosomal Enzyme Cathepsin L

“…Other residues in the vicinity of critical lysine residues may affect the accessibility or properties of the lysine residues. Effects such as this may explain the apparent involvement of tyrosine residues in phosphorylation of aspartylglucosaminidase and DNase I (16,29). In theory, any residue in contact with the transferase when it is bound to the protein could affect the rate and efficiency of phosphorylation.…”

“…Is the signal a complex, highly evolved structure that interacts with the transferase over an extended surface (29) or is it a relatively simple structure composed of a few well placed residues (17)? Two findings presented in this study address this issue.…”