ound at conventional positions in the primary structure. Approximately 10% of the kinome members have been classified as pseudokinases, only a handful of which have been rigorously studied. The POMK study underscores the importance of combining sequence analyses with structural characterization to judge the potential activity and function of these proteins. Most of the kinases residing in the cytosol and nucleus phosphorylate protein substrates. Our knowledge regarding glycan kinases remains in its infancy. Notably, among the thirteen secretory pathway kinases or kinase-like proteins discovered to date, two function as glycan kinases: Fam20B Zhu et al. eLife 2016;5:e22238. DOI: 10.7554/eLife.22238 11 of 18 Research article Biochemistry Biophysics and Structural Biology and POMK. Fam20B specifically recognizes the Gal-b4-Xyl disaccharide and phosphorylates the xylose residue, whereas POMK phosphorylates the mannose in the GalNAc-b3-GlcNAc-b4-Man trisaccharide. Both play critical roles to regulate glycan elongation: Fam20B for heparan sulfate and chondroitin sulfate proteoglycans, while POMK for a-DG. Phosphorylation of other types of extracellular glycans have been documented, whereas the responsible kinase remains elusive. MEK 162 Identifying the substrates of uncharacterized secretory kinases and understanding their function could shed light on the elaborate glycobiology in the secretory pathway and human physiology related to glycans. In summary, we have elucidated the molecular mechanism by which POMK catalyzes the phosphorylation of the trisaccharide GalNAc-b3-GlcNAc-b4-Man during the biosynthesis of functional aDG. Our study provides mechanistic insights into a unique `pseudokinase’ and a deeper understanding of the molecular mechanisms that underlie the pathogenesis of muscular dystrophy caused by POMK mutations. Materials and methods Protein expression and purification cDNA of HsPOMK and DrPOMK were purchased from Open Biosystems. cDNA of CoPOMK was prepared from the C. owczarzaki culture using the Trizol reagent and TransScript Reverse Transcriptase. For recombinant expression in insect cells, DNA fragments encoding HsPOMK, DrPOMK, and CoPOMK were cloned into the psMBP2 vector. Bacmids were generated using the Bac-to-Bac system. Recombinant baculoviruses were generated and amplified using the sf21 insect cells, maintained in the SIM SF medium. For protein production, Hi5 cells grown in the SIM HF medium were infected at a density of 1.52.0106 cells/ml. 48 hr post infection, 2 liters of conditioned medium were collected by centrifugation at 200 g. The medium was concentrated using a Hydrosart Ultrafilter and exchanged into the binding buffer containing 25 mM Tris-HCl, pH 8.0, 200 mM NaCl. The proteins were then purified using the Ni-NTA resin. Mutations were PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19825579 introduced into plasmids encoding HsPOMK by a PCR-based method, and the mutant proteins were purified similarly as the wild-type protein. For crystallization, TEV protease was used to remove the N-terminal 6xHis-MBP fusion tag from DrPOMK. Untagged DrPOMK was further purified by the anion exchange chromatography using a Resourse Q column, followed by the size-exclusion chromatography using a Superdex 200 16/60 column. To generate seleno-methionine labeled DrPOMK, Hi5 cells were adapted to a methioninefree medium and infected with baculovirus. 100 mg/L Se-Met was added to the medium at 12 and 36 hr post-infection. The Se-Met substituted protein was purified as described above. Product