TGN exit of the cation-independent mannose 6-phosphate receptor does not require acid hydrolase binding

Abstract

The cation-independent mannose 6-phosphate (Man-6-P) receptor (CI-MPR) binds newly synthesized, Man-6-P-containing lysosomal acid hydrolases in the trans-Golgi network (TGN) for clathrin-mediated transport to endosomes. It has remained unresolved, however, whether acid hydrolase binding is required for exit of the CI-MPR from the TGN. To address this question we used a B cell line derived from a Mucolipidosis type II (MLII)/I-cell disease patient. In MLII patients, acid hydrolases do not acquire the Man-6-P recognition marker and as a consequence do not bind to the CI-MPR. This causes secretion of the majority of the acid hydrolases and a decreased lysosomal activity resulting in typical inclusion bodies. In agreement herewith, ultrastructural analysis of the MLII patient derived B cells showed numerous inclusion bodies with undigested material, which we defined as autolysosomes. By quantitative immuno-electron microscopy we then studied the distribution of the CI-MPR in these cells. We found that the level of co-localization of TGN-localized CI-MPR and clathrin was similar in MLII and control B cells. Moreover, the CI-MPR was readily found in endosomes of MLII cells and the TGN-to-early endosome ratio of CI-MPR labeling was unaltered. These data show that there is no block in TGN exit of the CI-MPR in the absence of Man-6-P-modified acid hydrolases. Notably, late endosomes and inclusion bodies in MLII B cells contained increased levels of the CI-MPR, which likely reflects the reduced degradative capacity of these compartments.

Keywords:

• B cells
• cation-independent mannose 6-phosphate receptor
• electron microscopy
• lysosomal acid hydrolases
• mannose 6-phosphate modification
• Mucolipidosis type II
• trans-Golgi network

Abbreviations:

• CI-MPR, cation-independent MPR
• FBS, fetal bovine serum
• GA, glutaraldehyde
• IGF-II, insulin-like growth factor II
• MLII, Mucolipidosis type II
• MPR, mannose 6-phosphate receptor
• PB, phosphate buffer
• PFA, paraformaldehyde
• TGN, trans-Golgi network

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Acknowledgments

We thank the members of the Department of Cell Biology for helpful discussions and in particular Marc van Peski and René Scriwanek for their help with preparation of the electron micrographs, Dr Gillian Griffiths, Dr Peter Lobel and Dr Kurt von Figura for kindly providing the B cell lines, biotinylated sCI-MPR and CI-MPR antibody, respectively.

Additional information

Funding

This work was supported by VICI grant 918.56.611 of the Netherlands Organization for Scientific research (NWO) awarded to JK.