Novel synthetic biological study on intracellular distribution of human GlcNAc-1-phosphotransferase expressed in insect cells.

Many lysosomal enzymes contain N-glycans carrying mannose 6-phosphate (M6P) residues. Modifying lysosomal enzymes by M6P residues requires a two-step process in the Golgi apparatus. Then the lysosomal enzymes with M6P residues are transported from the trans-Golgi network to endosomes and lysosomes by M6P receptors. In insect cells, M6P residues are not added to N-glycans. Therefore, many insect lysosomal enzymes are transported to lysosomes by the M6P-independent pathway. The expression and subcellular distribution of M6P-modifying enzymes were examined by amplifying DNA fragments of M6P-modifying enzymes, generating the corresponding plasmid constructs, and transfection each construct into Sf9 cells, an insect cell line. The human GlcNac-1-phosphotransferase α/ß subunit, one of the M6P-modifying enzymes, was found to differ in maturation and localization between mammalian and insect cells. In mammalian cells, newly biosynthesized α/ß subunit localized in the cis-Golgi. In Sf9 cells, most of the α/ß subunit was localized in the endoplasmic reticulum, and few mature forms of α/ß subunit were observed. However, by the co-expression of the human site-1 protease, the mature forms were observed significantly and co-localization with each protein. Our study indicates new insights into regulating the intracellular distribution of the human GlcNac-1-phosphotransferase α/ß subunit in insect cells.

Glycan Recognition and Application of P-Type Lectins.

Cation-dependent mannose 6-phosphate receptor (CD-MPR) and cation-independent MPR (CI-MPR) belong to the P-type lectin family. Both intracellular and cell surface MPRs can recognize and bind with the terminal mannose 6-phospahte (M6P) residues of N-glycans attached to the mammalian lysosomal enzymes and the related co-factors. Domain9 (Dom9), which is one of the extracytoplasmic region of the CI-MPR, has relatively higher affinity for M6P residues. Here we describe the production of recombinant Dom9-His protein by Pichia pastris, purification, and application as a probe for lectin blotting.