TMEM2 is a bona fide hyaluronidase possessing intrinsic catalytic activity

• DOI: 10.1016/j.jbc.2023.105120
• Publication Date: 2023-07-30T13:44:54Z
• Authors: Takuma Narita; Yuki Tobisawa; Andrey Bobkov; Michael Jackson; Chikara Ohyama; Fumitoshi Irie; Yu Yamaguchi
• Keywords: Hyaluronidase; Ectodomain

Transmembrane protein 2 (TMEM2) was originally identified as a membrane-anchored of unknown function. We previously demonstrated that TMEM2 can degrade hyaluronan (HA). Furthermore, we showed induced global knockout Tmem2 in adult mice results rapid accumulation incompletely degraded HA bodily fluids and organs, supporting the identity cell surface hyaluronidase. In spite these advances, no direct evidence has been presented to demonstrate intrinsic hyaluronidase activity TMEM2. Here, directly establish catalytic The ectodomain (TMEM2ECD) expressed His-tagged soluble purified by affinity size-exclusion chromatography. Both human mouse TMEM2ECD robustly fluorescein-labeled into 5 10 kDa fragments. exhibits this HA-degrading irrespective species origin position epitope tag insertion. is more potent than HYAL2, which, like TMEM2, implicated degradation. Finally, show not only but also native high-molecular weight HA. addition core findings, our study reveals hitherto unrecognized confounding factors, such quality reagents choice assay systems, could lead erroneous conclusions regarding conclusion, legitimate functional Our findings raise cautions methods for performing degradation assays hyaluronidases. Hyaluronan (HA) an extremely large polysaccharide glycosaminoglycan family, representing one major components extracellular matrices. Via its unique biochemical biophysical properties, plays key roles regulating size, fluid pressure, malleability tissues. small fragments exert range effects on behavior signaling (1Toole B.P. Hyaluronan: from glue pericellular cue.Nat. Rev. Cancer. 2004; 4: 528-539Crossref PubMed Scopus (1746) Google Scholar). vivo, continuously turned over with short half-life. 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As Figure 1C, completely 6 h, bulk reaction occurring within h. Analysis chromatography yielded similar result 1D). These possesses Significantly, occurs much rapidly observed cell-based TMEM2-transfected 293T (∼72 h; Scholar)), system employed discrepancy rates suggests influenced component(s) suppresses (see below data: Factors obtained assays). substantiate initial finding, four corroborating experiments systems. First, same structural configuration TMEM2ECD, robust 2A). time course faster being almost complete 1 Second, examined possible effect TMEM2ECD. For this, N terminus. N-terminally tagged nearly C-terminally 2B). Third, full-length associated membranes isolated membrane fraction source. both 2C). seen Fig. 2A), stronger system. activities further confirmed situ 293 2D). 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Another possibility differences labeling condition denatured resistant treatment any event, emphasize needs re-scrutinize earlier questionable products. contradictory Sato (20Sato although exhibited assay. authors claim (in contrast TMEM2) noted Results might factor(s) analyzed TMEM2ECD-transfected cells. little detected TMEM2ECD-containing supernatant, noticeable emerges upon 30-fold concentration supernatant 50 cutoff spin column 4B). Remarkably, dilution concentrated regular buffer emergence highly strongly suppressed under conditions assays, indeed possess suppression must mediated substance(s) passes through then reduced insignificant levels dilution. At present, Discussion). report identifying extensive illustrating significance Yet, left uncertainty about led speculation rather act accessory regulatory molecule known convincingly paper include without requiring participation proteins; assays; HYAL2; (iv) should stressed contrary recent lacks clearly somewhat less active assays. 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