Page 20 - T. Protein modification and regulation

[T. Protein modification and regulation-13]

Enhanced mannosylphosphorylation for high-mannose type

N-glycan by in vitro reaction of recombinant Mnn14

Na-young Jung¹, Ji-Yeon Kang¹, Doo-Byoung Oh¹

¹Environmental Disease Research Center, KRIBB, Deajeon 34141, Korea

lysosomal storage diseases (LSDs) is caused by a deficiency of the lysosomal enzyme that leads to accumulation of
undigested substrates. LSDs is currently treated by enzyme replacement therapy (ERT) with recombinant enzymes,
which is mediated by mannose-6-phosphate receptors (MPRs) on plasma membrane for targeting to lysosomes.

Therefore, the content of mannose-6-phosphate is an important quality factor of therapeutic enzymes for lysosomal

delivery. In Saccharomyces cerevisiae yeast, MNN4 and MNN6 genes are required for mannosylphosphorylation.
Currently, we found that, MNN14 gene having homology with MNN4 mediates mannosylphosphorylation and
disruption of MNN14 gene abolished N-glycan mannosylphosphorylation. In this study, we recombinantly expressed

Mnn14 in pichia pastoris which are constructed by deleting 30-amino acid, 60- amino acid, 100- amino acid in N-
terminal region or 14-amino acid, 85- amino acid in C-terminal region. The activity of mannosylphosphorylation

was analyzed by HPLC and DNA sequencer using a high-mannose type N-glycans (Man8) and GDP-mannose as an
acceptor and donor substrates. The analyzed results show that d30rM14 has highest avtivity compare to the other

Mnn14s and it was able to mannosylphosphorylate the high mannose-type N-glycans of rhGAA. The recombinant
Mnn14 having strong mannosylphosphorylation activity shows promise for the generation of therapeutic enzymes

with high content of mannose-6-phosphate glycans and improved lysosomal targeting capability.