MARCH1 has emerged as a critical regulator of antigen presentation, serving to suppress the ability of immature APC to activate T cells through its effects on MHC and costimulatory molecules. The transition of APC from the immature to the mature state is essential for effective T cell priming by APC, and is necessarily subject to tight regulation. Though MARCH1 is clearly a component of this regulatory system, much remains to be determined regarding its involvement and control. MARCH1 transcription decreases in response to DC maturation with LPS (13
), and increases after stimulation of monocytes with the anti-inflammatory cytokine IL-10 (21
). Thus, MARCH1 activity is regulated, at least in part, through changes in gene transcription. Since MARCH1 protein levels appear to be balanced such that relatively small changes significantly alter the cell surface display of MHC class II, then the instability of MARCH1 is an essential component of its regulation. Even in immature DC where MARCH1 is active, MARCH1 turns over quite rapidly. It should be noted that we have not observed any appreciable increase in MARCH1 turnover following maturation with LPS (data not shown), which is known to enhance lysosomal activity in DC (60
). It is interesting that the more stable N-terminal mutants appear to be somewhat more potent than wildtype MARCH1 at downregulating CD86 (), consistent with the idea that the inherent stability of MARCH1 maintains its levels at a critical functional threshold.
Lysosomal acidification significantly affects the levels of MARCH1, and this implies proteolysis of MARCH1 within endo-lysosomal compartments, where MHC class II ubiquitination appears to occur (11
). However, the topology of MARCH1 presents a potential obstacle to this model. MARCH1 possesses two transmembrane domains, with a short, connecting lumenal domain; the bulk of the molecule resides on the cytosolic face of the membrane (16
). How could lysosomal enzymes access MARCH1? It is possible that the lumenal domain is targeted, but replacement of this domain with unrelated sequences did not stabilize MARCH1 (not shown). Further, the size of the fragments of MARCH1 which we detect is not consistent with cleavage within the lumenal domain (data not shown). The most likely explanation is that the cytosolic domains of MARCH1 are exposed to proteases within multivesicular bodies (61
). Indeed, electron microscopy has shown an abundance of MHC class II molecules within these structures (3
), and this requires ubiquitination of the MHC class II beta-chain (11
). Therefore, it is probable that MARCH1 also traffics through multivesicular bodies where it is exposed to the myriad proteases present in this compartment. Among these proteases, cathpesins are likely candidates to affect MARCH1, given their established roles in antigen processing (3
). Most of the cathepsins are cysteine proteases (46
) and we found that the cysteine/serine protease inhibitor leupeptin was able to stabilize MARCH1. Further, inhibition of cathepsin L increased MARCH1 levels through stabilization, but not in all cell types. We failed to observe any stabilizing effect of cathepsin S inhibition/knockdown on MARCH1. From our results, we conclude that MARCH1 protein levels are regulated by multiple, redundant proteases within lysosomes, including cathepsin L. A similar finding has emerged for the processing of Toll-like Receptor 9, which must be cleaved within endo-lysosomes prior to signaling, and this cleavage event can be mediated by different proteases (62
Our characterization of mutant forms of MARCH1 complemented our studies of the factors which affect MARCH1 protein levels. In particular, we identified a region of MARCH1 which influenced its stability. Removal of as few as the first 40 residues of the N-terminus affected the stability of MARCH1, without compromising activity. The increased stability of the N-terminal truncations was correlated with a change in the subcellular distribution of the mutants relative to wildtype. Most notably, in addition to the Golgi and lysosomal staining seen with wildtype MARCH1, these mutants exhibited abundant vesicular staining throughout the cell. The distribution of MARCH1 raises questions about its subcellular site of action. At steady-state, we observed the most pronounced co-localization between MARCH1 and a trans
-Golgi marker, with relatively little MARCH1 present within endo-lysosomes. However, since MARCH1 turnover requires lysosomal activity, it must traffic through this compartment. In the case of MHC class II, it seems that the effects of MARCH1 are manifested in a post-Golgi compartment, after processing of the invariant-chain (11
), resulting in rapid endocytosis of MHC class II from the cell surface (12
). Consistent with this finding, it was shown that human MARCH1 can be detected in early endosomes (transferrin-receptor-positive), but not later, HLA-DM-positive compartments (13
). In this instance, MARCH1 was expressed as a fusion with GFP, which could affect its localization, perhaps causing some accumulation in early endosomes where it could continue to ubiquitinate MHC class II. Though MHC class II molecules are quickly internalized in the presence of MARCH1, it is possible that ubiquitination occurs prior to the initial arrival of MHC class II at the cell surface or during recycling within early endosomes. Upon reaching the cell surface, ubiquitin could then exert its effects on MHC class II. Whatever the case, MARCH1 itself appears to traffic through the late endocytic compartment, at least transiently, in order to be degraded.
Collectively, the available data suggest a dynamic pattern of trafficking along the endocytic pathway for MARCH1, between the trans
-Golgi, early endosomes, and late endosomes. Our results indicate that this trafficking requires information within the N-terminus. However, disruption of this pathway does not necessarily abolish function. Rather, it affects the stability of MARCH1, which appears to be important for maintaining the proper levels of MARCH1. Deletion of the C-terminal 50 residues (ΔC229-279
) did not dramatically affect localization or stability of MARCH1. Notably, this mutant retains both of the C-terminal YXXΦ motifs (). However, mutation of these motifs affected MARCH1 function, but not expression levels, arguing that they are not required for trafficking into endo-lysosmes. Rather, these residues may be important for substrate interaction or recruitment of downstream effector molecules. While it remains to be determined exactly where and when MHC class II and CD86 encounter MARCH1 during biogenesis, and how this process is influenced by APC maturation, it seems likely that these two targets of MARCH1 share some common steps in their trafficking. This may help explain how these two unrelated proteins can both be targeted by the same E3 ligase. A curious feature of the viral RING-CH E3 ligases is the ability of some to target multiple, unrelated substrates (18
). As a whole, the basis of substrate recognition by viral RING-CH molecules is not well understood. Our characterization of the viral mK3 protein has demonstrated a clear requirement for “adapter-type” proteins in substrate recruitment (31
). MARCH1 may also require cofactors to assist in recruitment of its distinct substrates, and such cofactors would likely tie into the trafficking pathways utilized by MARCH1 and its substrates. Elucidation of the full spectrum of molecules involved in MARCH1-dependent regulation of antigen presentation will be essential to understand how MARCH1 may contribute to both immune activation and tolerance.