The safety, tolerability and preliminary efficacy of mannose 6-phosphate in enhancing the outcome in Zone II flexor tendon repair was studied in a multicentre parallel double-blinded randomized controlled trial. Eight UK teaching hospitals were involved in treating repaired flexor tendons with a single intraoperative intrathecal dose of 600 mM mannose 6-phosphate, with follow-up over 26 weeks. A total of 39 patients (mannose 6-phosphate, n = 20; standard care, n = 19) were randomized. Seven were excluded from the safety and tolerability analysis because of intraoperative findings and eight were excluded due to early dropout (n = 4) or tendon rupture (n = 4), leaving 24 (mannose 6-phosphate, n = 13; standard care, n = 11) for assessment of total active motion. The safety, tolerability and other side effects were comparable between the groups. There was no significant difference between the two groups in the total active motion at Week 26. We concluded that mannose 6-phosphate, although safe and tolerable, had no beneficial effect on finger range of motion after Zone II tendon division.Level of evidence 1b.

As most recombinant lysosomal enzymes are incorporated into cells via mannose 6-phosphate (M6P) receptors, the M6P content is important for effective enzyme replacement therapy (ERT) for lysosomal diseases. However, there have been no comprehensive reports of the M6P contents of lysosomal enzymes. We developed an M6P assay method comprising three steps, i.e., acid hydrolysis of glycoproteins, derivatization of M6P, and high-performance liquid chromatography, and determined the M6P contents of six recombinant lysosomal enzymes now available for ERT and one in the process of development. The assay is easy, specific, and reproducible. The results of the comparative study revealed that the M6P contents of agalsidase alfa, agalsidase beta, modified α-N-acetylgalactosaminidase, alglucosidase alfa, laronidase, idursulfase, and imiglucerase are 2.1, 2.9, 5.9, 0.7, 2.5, 3.2, and <0.3 mol/mol enzyme, respectively. The results were correlated with those of the biochemical analyses previously performed and that of the binding assay of exposed M6P of the enzymes with the domain 9 of the cation-independent M6P receptor. This assay method is useful for comparison of the M6P contents of recombinant lysosomal enzymes for ERT.

Phosphomannose isomerase is a zinc metalloenzyme that catalyzes the reversible isomerization of mannose-6-phosphate and fructose-6-phosphate, and the three-dimensional (3D) structure of human phosphomannose isomerase has not been reported. In order to understand the catalytic mechanism, the 3D structure of the protein is built by using homology modeling based on the known crystal structure of mannose-6-phosphate isomerase from (PDB code 1PMI). The model structure is further refined by energy minimization and molecular dynamics methods. The mannose-6-phosphate-enzyme complex is developed by molecular docking and the key residues involved in the ligand binding are determined, which will facilitate the understanding of the action mode of the ligands and guide further genetic studies. Our results suggest a hydride transfer mechanism of alpha-hydrogen between the C1 and C2 positions but do not support the cis-enediol mechanism. The detailed mechanism involves, on one side, Zn2+ mediating the movement of a proton between O1 and O2, and, on the other side, the hydrophobic environment formed in part by Tyr278 promoting transfer of a hydride ion.

The binding and internalization of a model lysosomal enzyme, beta-galactosidase, was visualized by use of rabbit anti-beta-galactosidase and goat anti-rabbit IgG; the second antibody was labeled with rhodamine or fluorescein (for detection by fluorescence) or with horseradish peroxidase (for electron microscopy). Chinese hamster ovary cells were incubated with beta-galactosidase at 4 degrees C, and then were washed and sequentially incubated in the cold with the two antibodies. The beta-galactosidase was found primarily in coated pits. The binding of the enzyme was completely inhibited by 5 mM mannose 6-phosphate. After the reaction with enzyme and antibodies, the cells were warmed to 37 degrees C; within 1 minute, the beta-galactosidase--antibody complex had begun to move to uncoated vesicles (receptosomes). After 8 min, the beta-galactosidase--antibody complex was seen in receptosomes near tubular elements in the Golgi/GERL area, within such tubular elements and at times, in vesicular elements that may correspond to coated structures of the GERL system. After 15 min, the enzyme--antibody complex was found in lysosomes near the Golgi/GERL are and a half-hour later it was in lysosomes distributed throughout the cytoplasm. Double-label experiments using beta-galactosidase and gold/alpha 2-macroglobulin showed the presence of the two ligands in the same coated pits and receptosomes. Thus, the pathway for internalization of beta-galactosidase via the mannose 6-phosphate receptor is similar to the pathway established for other ligands such as low density lipoprotein and alpha 2-macroglobulin.

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