Role of N-linked oligosaccharide flexibility in mannose phosphorylation of lysosomal enzyme cathepsin L.
Mannose phosphorylation of N-linked oligosaccharides by UDP-GlcNAc:lysosomal enzyme N-acetylglucosamine-1-phosphotransferase is a key step in the targeting of lysosomal enzymes in mammalian cells and tissues. The selectivity of this process is determined by lysine-based phosphorylation signals shared by lysosomal enzymes of diverse structure and function. By introducing new glycosylation sites at several locations on the surface of mouse procathepsin L and modeling oligosaccharide conformations for sites that are phosphorylated, it was shown that the inherent flexibility of N-linked oligosaccharides can account for the specificity of the transferase for oligosaccharides at different locations on the protein. By using this approach, the physical relationship between the lysine-based signal and the site of phosphorylation of mannose residues was determined. The analysis also revealed the existence of additional independent lysine-based phosphorylation signals on procathepsin L, which account for the low level of phosphorylation observed when the primary Lys-54/Lys-99 signal is ablated. Mutagenesis of residues that surround Lys-54 and Lys-99 and demonstration of mannose phosphorylation of a glycosylated derivative of green fluorescent protein provide strong evidence that the cathepsin L phosphorylation signal is a simple structure composed of as few as two well placed lysine residues.
Warner
Jason B
JB
Department of Physiology, Tufts University School of Medicine, Boston, Massachusetts 02111, USA.
Thalhauser
Craig
C
Tao
Kai
K
Sahagian
G Gary
GG
D000818:Animals; D019556:COS Cells; D056668:Cathepsin L; D002403:Cathepsins / Q000737:chemistry* / Q000378:metabolism; D003546:Cysteine Endopeptidases; D006031:Glycosylation; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000378:metabolism; D008247:Lysosomes / Q000201:enzymology*; D008358:Mannose / Q000378:metabolism*; D009844:Oligosaccharides / Q000737:chemistry*; D010766:Phosphorylation
D008164:Luminescent Proteins; D009844:Oligosaccharides; D049452:Green Fluorescent Proteins; D002403:Cathepsins; D003546:Cysteine Endopeptidases; D056668:Cathepsin L; C534278:Ctsl protein, mouse; D008358:Mannose