A collagen glucosyltransferase drives lung adenocarcinoma progression in mice

Abstract

Cancer cells are a major source of enzymes that modify collagen to create a stiff, fibrotic tumor stroma. High collagen lysyl hydroxylase 2 (LH2) expression promotes metastasis and is correlated with shorter survival in lung adenocarcinoma (LUAD) and other tumor types. LH2 hydroxylates lysine (Lys) residues on fibrillar collagen’s amino- and carboxy-terminal telopeptides to create stable collagen cross-links. Here, we show that electrostatic interactions between the LH domain active site and collagen determine the unique telopeptidyl lysyl hydroxylase (tLH) activity of LH2. However, CRISPR/Cas-9-mediated inactivation of tLH activity does not fully recapitulate the inhibitory effect of LH2 knock out on LUAD growth and metastasis in mice, suggesting that LH2 drives LUAD progression, in part, through a tLH-independent mechanism. Protein homology modeling and biochemical studies identify an LH2 isoform (LH2b) that has previously undetected collagen galactosylhydroxylysyl glucosyltransferase (GGT) activity determined by a loop that enhances UDP-glucose-binding in the GLT active site and is encoded by alternatively spliced exon 13 A. CRISPR/Cas-9-mediated deletion of exon 13 A sharply reduces the growth and metastasis of LH2b-expressing LUADs in mice. These findings identify a previously unrecognized collagen GGT activity that drives LUAD progression.

Comparative genomic and crystal structure analyses identify a collagen glucosyltransferase from Acanthamoeba Polyphaga Mimivirus

“…2F and supplemental Fig. 1) (12). Under these conditions, R699 showed robust activity with UDP-Glc but no other sugar donors ( Fig.…”

“…The largest 14-residue deletion ( Fig. 4E brown shallow) happens in the accessory domain that is not required for LH3’s GGT activity but modulates LH2’s GGT activity (12,46). This largest deletion shortens 2 alpha helices and 1 beta strand, resulting in a major conformation change (curved arrow in Fig.…”

“…GGT activity was measured similarly as previously described (12). The assay was performed in reaction buffer (100 mM HEPES buffer pH 8.0, 150 mM NaCl) at 37 °C for 1 h with 1 μM R699 enzyme, 100 μM MnCl 2 , 200 μM UDP-Glc (MilliporeSigma, St. Louis, MO), 1 mM dithiothreitol and 1.75 mM galactosyl hydroxylysine (Gal-Hyl, Cayman Chemical, Ann Arbor, MI) or 2 μM deglucosylated collagen IV.…”

“…It is generally accepted that LH1 is the main LH for the helical domain and LH2 for the telopeptides. Certain Hyl residues in the collagen helical domain are galactosylated by glycosyltransferase 25 domain containing 1 and 2 (GLT25D1 and GLT25D2) and then glucosylated by lysyl hydroxylases to form a unique Hyl-O-linked glycosylation with a mono-or di-saccharide (7)(8)(9)(10)(11)(12). LH3 is the major galactosylhydroxylysyl glucosyltransferase (GGT) catalyzing collagen glucosylation (8), although it has been shown to be a multifunctional enzyme (13).…”

“…Since the initial release of the mimiviral genome (28), studies have identified 7 mimiviral collagen genes and 2 mimiviral collagen-modifying enzyme genes that encode three enzymes, including collagen prolyl hydroxylase, collagen lysyl hydroxylase, and collagen hydroxylysyl glucosyltransferase (26,29). Structural and functional studies of mimiviral collagen lysyl hydroxylase provide insights into functions of the human collagen-modifying enzymes (12,30). Since collagen is widely used for tissue and biomaterial engineering, efforts have been made to express recombinant collagens using different expression systems (31)(32)(33).…”