Structure of the transmembrane protein 2 (TMEM2) ectodomain and its apparent lack of hyaluronidase activity

Abstract

Background: Hyaluronic acid (HA) is a major polysaccharide component of the extracellular matrix. HA has essential functions in tissue architecture and the regulation of cell behaviour. HA turnover needs to be finely balanced. Increased HA degradation is associated with cancer, inflammation, and other pathological situations. Transmembrane protein 2 (TMEM2) is a cell surface protein that has been reported to degrade HA into ~5 kDa fragments and play an essential role in systemic HA turnover. Methods: We produced the soluble TMEM2 ectodomain (residues 106-1383; sTMEM2) in human embryonic kidney cells (HEK293) and determined its structure using X-ray crystallography. We tested sTMEM2 hyaluronidase activity using fluorescently labelled HA and size fractionation of reaction products. We tested HA binding in solution and using a glycan microarray. Results: Our crystal structure of sTMEM2 confirms a remarkably accurate prediction by AlphaFold. sTMEM2 contains a parallel β-helix typical of other polysaccharide-degrading enzymes, but an active site cannot be assigned with confidence. A lectin-like domain is inserted into the β-helix and predicted to be functional in carbohydrate binding. A second lectin-like domain at the C-terminus is unlikely to bind carbohydrates. We did not observe HA binding in two assay formats, suggesting a modest affinity at best. Unexpectedly, we were unable to observe any HA degradation by sTMEM2. Our negative results set an upper limit for kcat of approximately 10-5 min-1. Conclusions: Although sTMEM2 contains domain types consistent with its suggested role in TMEM2 degradation, its hyaluronidase activity was undetectable. HA degradation by TMEM2 may require additional proteins and/or localisation at the cell surface.

PKHD1L1, a gene involved in the stereocilia coat, causes autosomal recessive nonsyndromic hearing loss

“…S4 ) (p.His552 in Hs TMEM2, Fig. 4 a; Protein Data Bank (PDB): 8C6I (Niu et al 2023 )) reported to form a nickel-finger binding site, which might mediate catalytic functions in TMEM2. Disruption of this site in PKHD1L1 and TMEM2 might impair cation binding (Fig.…”

“…S3 for full PKHD1L1 sequence alignment). Residue numbering for TMEM2 as in PDB: 8C6I (Niu et al 2023 ), while residue numbering for Hs PKHD1L1 as in NCBI accession code NP_803875.2 with the signal peptide included (Supplementary Table S1 , 26 residues are suggested according to protein sequence alignment, see Methods). Green triangles point to the location of the Hs p.(His2479Gln) variant, orange circles ( left ) indicate 100% amino acid sequence identity for this PKHD1L1 fragment between the Hs , Pt , and Mm2 species (See supplementary Table S1 for details about the selected orthologs).…”

PKHD1L1, A Gene Involved in the Stereocilia Coat, Causes Autosomal Recessive Nonsyndromic Hearing Loss

“…S4) (p.His552 in Hs TMEM2, Fig. 4a; Protein Data Bank (PDB): 8C6I (Niu et al 2023)) reported to form a nickel-finger binding site, which might mediate catalytic functions in TMEM2. Disruption of this site in PKHD1L1 and TMEM2 might impair cation binding (Fig.…”

“…TableS1for details of the selected species, Supplementary Fig.S4for sequence alignment of this specific fragment, and Supplementary Fig.S3for full PKHDL1 sequence alignment). Residue numbering for TMEM2 as in PDB: 8C6I(Niu et al 2023), while residue numbering for Hs PKHD1L1 as in NCBI accession code NP_803875.2 with the signal peptide included (Supplementary TableS1, 26 residues are suggested according to protein sequence alignment, see Methods). Green triangles point to the location of the Hs p.(His2479Gln) variant, orange circles (left) indicate 100% amino acid sequence identity for this PKHD1L1 fragment between the Hs, Pt, and Mm2 species (See supplementary Table…”

The molecular structure and function of fibrocystin, the key gene product implicated in autosomal recessive polycystic kidney disease (ARPKD)

“…The crystal structure of the TMEM2 ectodomain. (a) Overview of the structure (PDB: 8C6I) with domains colour‐coded and labelled (Niu et al., 2023). Fibrocystin is predicted to have two G8 domains, each followed by parallel β‐helical repeats, possibly folding into a similar conformation as those of TMEM2.…”