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Targeted protein transport: Show your sugar pass

Mannose 6-phosphorylation of cystatin F glycans is necessary for its internalization and activation from extracellular pools. Immunofluorescence image provided by J Colbert. Cystatin F: red, lysosomes: green.

Cystatins are cysteine protease inhibitors, whose targets include the lysosomal proteases cathepsins. Class II cystatins, including cystatin F, are made using a signal sequence and secreted into the extracellular space, where it is thought they suppress potentially damaging cathepsin activity that has escaped the lysosomal system. Unusually, cystatin F is partially retained in the endocytic system and, although also secreted, is only active inside the cell. It is not yet known how endosomal targeting of cystatin F is achieved, or why it is also secreted in inactive form. Now in Traffic, Colbert et al have shown using immune cells that N-linked oligosaccharides divert cystatin F to the endocytic pathway, both within the originating cell and into neighbouring, non-expressing cells.

Uniquely among cystatins, cystatin F is initially made as an inactive disulfide-linked dimer. It is expressed primarily in immune cells and is one of just two glycosylated cystatins. The active monomeric form is produced in the endocytic pathway by proteolytic cleavage. Colbert et al used cystatin F mutants to show that dimerization is not necessary for diversion from the secretory to the endocytic pathway, but this targeting instead relies on N-glycosylation. Glycosylation of Asn62, which occurs in the usual glycosylation recognition sequence Asn-X-Ser/Thr, sufficed to specify endosomal sorting. Interestingly, when this glycosylation was blocked by mutation, targeting was instead achieved by glycosylation of a 'back-up' site on Asn61 using the less common sequence Asn-X-Cys. The EndoH-sensitive incorporation of radiolabelled orthophosphate indicated that cystatin F glycosylation is modified in the Golgi to include mannose-6-phosphate (M6P), which is recognised by the M6P receptors that transport many lysosomal hydrolases. With the use of knockout cell lines, the authors showed that endosomal/lysosomal retention of cystatin F requires the cation-independent M6P receptor (CI-MPR).

In addition to intracellular lysosomal targeting, CI-MPR is also found at the cell surface where it mediates internalization of extracellular M6P-tagged enzymes. Using conditioned medium from cells transfected with wild type and mutant cystatin F, Colbert et al showed that secreted, glycosylated cystatin F is taken up by non-expressing cells and this can be blocked by either CI-MPR knockout or increasing concentrations of M6P. Unlike intracellular transport, internalization of cystatin F requires the dimeric form, presumably due to increased avidity for the receptor from the double glycosylation. Importantly, the internalized cystatin F is activated in recipient cells, indicated by the suppression of endogenous cathepsin C activity, a result that was replicated in primary immune cells.

Immune cells secreting cystatin F may be able to regulate protease activity in neighboring cells that do not express the inhibitor or do at insufficient levels. The production of an inactive dimer ensures that activity is saved for the correct compartments and should allow efficient intercellular transfer unimpeded by substrate binding. Glycosylation targets cystatin F to the endocytic pathway but incomplete retention in the originating cell results in its secretion, allowing paracrine uptake using the same glycan signal. Attention is now turning to whether the activating proteolytic cleavage of cystatin F is achieved by the same enzymes as it inhibits.

Author: Emma Leah