Fig. 2: LH2 t-LH specificity is determined by electrostatic interactions with collagens.

a Design of LH constructs. Glycosyltransferase (GLT), accessory (AC), and lysyl hydroxylase (LH) domains. LH2’s LH domain replaced with that of LH1 (LH2/1). b LH2 protein levels were determined by western blot analysis of MC-3T3 (MC) cells that were stably co-transfected with LH2 shRNA (shLH2) and empty vector (-) or vectors expressing LH2, LH1, or LH2/1. Actin used as loading control. c–g Collagen cross-link quantification of matrices derived from MC-3T3 (MC) cells described in (b). Dihydroxylysinonorleucine (DHLNL, c), pyridinoline (Pyr, d), histidinohydroxymerodesmosine (HHMD, e), hydroxylysinonorleucine (HLNL, f), and the HLCC-to-LCC ratio (g). The HLCC-to-LCC ratio was calculated as (DHLNL + Pyr)/HHMD. h LH2b LH domain structure was modeled using a homology-modelling server SWISS-MODEL. The structure model identifies a cluster of arginine residues (marine) near the LH2 active site. Fe²⁺ molecule (orange ball) in active site. i Amino acid sequence alignment of human LHs. R680 and R682 in LH2 (arrow heads) are not in LH1 and LH3. j–m LH activity of LH2 and LH2-EP on a trimeric telopeptide (j, l, m) or type I collagen (k). Acidic residues at i-1 and i-2 positions in telopeptide (Telo) were mutated to alanine (TeloAA). LH activity was measured by detecting succinate production with an adenosine triphosphate (ATP)-based luciferase assay. n and o Numbers of visible primary lung tumors (left plot) and metastases to mediastinal nodes (middle plot) or contralateral lung (right plot). Mice were injected intra-thoracically with parental (WT) or CRISPR/Cas-9-edited 344SQ cells. Plod2 mutations ablate LH2’s tLH activity owing to loss of dimerization (L735D) (n) or Fe²⁺-binding (D689A) (o). Results are mean values (±S.D.) from replicate samples. For (c–g) and (j–m), n = 3. For (n and o), n = 9 or 10. Error bars indicate ±S.D. p values, 2-tailed Student’s t test.