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B.S., Peking University – 2006
Thesis Advisor: Peter Lobel, Ph.D.
Graduate Program in Cellular &
CABM Room 010
Monday, April 18, 2011
Lysosomes are membrane-delimited organelles that are essential for degradation of macromolecules. Most newly-synthesized lysosomal proteins reach lysosomes via the mannose 6-phosphate (Man6-P) targeting pathway. Upon reaching lysosome, the Man6-P modification is quickly removed in most cell types. However, the enzyme responsible for removing the Man6-P recognition marker has remained elusive.
In the first part of this thesis, I identified acid phosphatase 5 (ACP5) as a candidate for the enzyme that removes Man6-P modification. Two pieces of evidence suggest that ACP5 is responsible for this phosphatase activity: 1) expression of ACP5 in N1E-115 neuroblastoma cells, which do not efficiently dephosphorylate lysosomal proteins, significantly decreased the steady state levels of Man6-P glycoproteins; 2) deficiency in ACP5 caused highly elevated levels of Man6-P glycoproteins in tissues that normally express ACP5.
These results indicate a central role for ACP5 in removal of the Man6-P modification and lead us to investigate the biotechnological application of ACP5 in identifying lysosomal protein candidates and improving therapeutic protein production for lysosomal storage diseases (LSDs).
Deficiencies in lysosomal proteins can cause LSDs. But for some LSDs, the molecular mechanism remains unknown. Identifying new lysosomal proteins can provide candidates for LSDs and further deepen our understanding of lysosomal biology.
In the second part of my thesis, I used ACP5-deficient tissues as a rich source of Man6-P glycoproteins to search for new lysosomal proteins. Six different ACP5 deficient mouse tissues were subjected to affinity chromatography on immobilized Man6-P receptors. Fractions from a “mock” elution and a “specific” elution were analyzed by mass spectrometry and statistical methods were used to distinguish Man6-P glycoproteins from contaminants. This analysis yielded 63 known lysosomal proteins and an additional 66 lysosomal protein candidates.
A number of Man6-P containing lysosomal proteins are currently approved for treatment of different LSDs. In the third part of my thesis, I explored the possibility of using milk from ACP5 deficient animals to improve production of phosphorylated lysosomal proteins. Affinity purification of Man6-P glycoproteins in milk from wild-type and ACP5-deficient mice and from wild-type cow indicate that over 40 lysosomal proteins are secreted into milk. In addition, milk lysosomal proteins tend to be highly Man6-phosphorylated (~20-30%) comparing to other tissues (<1% in liver). However, deficiency in ACP5 does not increase the Man6-P glycoprotein levels. These results indicate that transgenic animals could be useful for production of lysosomal therapeutic proteins but that further engineering of animals to inactivate ACP5 would be of limited utility.