TMEM2 is a bona fide hyaluronidase possessing intrinsic catalytic activity

Transmembrane protein 2 (TMEM2) was originally identified as a membrane-anchored protein of unknown function. We previously demonstrated that TMEM2 can degrade hyaluronan (HA). Furthermore, we showed that induced global knockout of Tmem2 in adult mice results in rapid accumulation of incompletely degraded HA in bodily fluids and organs, supporting the identity of TMEM2 as a cell surface hyaluronidase. In spite of these advances, no direct evidence has been presented to demonstrate the intrinsic hyaluronidase activity of TMEM2. Here, we directly establish the catalytic activity of TMEM2. The ectodomain of TMEM2 (TMEM2^ECD) was expressed as a His-tagged soluble protein and purified by affinity and size-exclusion chromatography. Both human and mouse TMEM2^ECD robustly degrade fluorescein-labeled HA into 5 to 10 kDa fragments. TMEM2^ECD exhibits this HA-degrading activity irrespective of the species of TMEM2 origin and the position of epitope tag insertion. The HA-degrading activity of TMEM2^ECD is more potent than that of HYAL2, a hyaluronidase which, like TMEM2, has been implicated in cell surface HA degradation. Finally, we show that TMEM2^ECD can degrade not only fluorescein-labeled HA but also native high-molecular weight HA. In addition to these core findings, our study reveals hitherto unrecognized confounding factors, such as the quality of reagents and the choice of assay systems, that could lead to erroneous conclusions regarding the catalytic activity of TMEM2. In conclusion, our results demonstrate that TMEM2 is a legitimate functional hyaluronidase. Our findings also raise cautions regarding the choice of reagents and methods for performing degradation assays for hyaluronidases.