Lysyl hydroxylase 2 mediated collagen post-translational modifications and functional outcomes

Masahiko Terajima; Yuki Taga; Tomoyuki Nakamura; Hou Fu Guo; Yukako Kayashima; Nobuyo Maeda-Smithies; Kshitij Parag-Sharma; Jeong Seon Kim; Antonio L. Amelio; Kazunori Mizuno; Jonathan M. Kurie; Mitsuo Yamauchi

doi:10.1038/s41598-022-18165-0

Lysyl hydroxylase 2 mediated collagen post-translational modifications and functional outcomes

Masahiko Terajima, Yuki Taga, Tomoyuki Nakamura, Hou Fu Guo, Yukako Kayashima, Nobuyo Maeda-Smithies, Kshitij Parag-Sharma, Jeong Seon Kim, Antonio L. Amelio, Kazunori Mizuno, Jonathan M. Kurie, Mitsuo Yamauchi

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Abstract

Lysyl hydroxylase 2 (LH2) is a member of LH family that catalyzes the hydroxylation of lysine (Lys) residues on collagen, and this particular isozyme has been implicated in various diseases. While its function as a telopeptidyl LH is generally accepted, several fundamental questions remain unanswered: 1. Does LH2 catalyze the hydroxylation of all telopeptidyl Lys residues of collagen? 2. Is LH2 involved in the helical Lys hydroxylation? 3. What are the functional consequences when LH2 is completely absent? To answer these questions, we generated LH2-null MC3T3 cells (LH2KO), and extensively characterized the type I collagen phenotypes in comparison with controls. Cross-link analysis demonstrated that the hydroxylysine-aldehyde (Hyl^ald)-derived cross-links were completely absent from LH2KO collagen with concomitant increases in the Lys^ald-derived cross-links. Mass spectrometric analysis revealed that, in LH2KO type I collagen, telopeptidyl Lys hydroxylation was completely abolished at all sites while helical Lys hydroxylation was slightly diminished in a site-specific manner. Moreover, di-glycosylated Hyl was diminished at the expense of mono-glycosylated Hyl. LH2KO collagen was highly soluble and digestible, fibril diameters were diminished, and mineralization impaired when compared to controls. Together, these data underscore the critical role of LH2-catalyzed collagen modifications in collagen stability, organization and mineralization in MC3T3 cells.

Original language	English
Article number	14256
Journal	Scientific Reports
Volume	12
Issue number	1
DOIs	https://doi.org/10.1038/s41598-022-18165-0
State	Published - Dec 2022

Bibliographical note

Publisher Copyright:
© 2022, The Author(s).

Funding

This work was supported by NIH R01CA251067 and SPORE P50CA070907 to JMK and MY, Basic Science Fund from Nippi to YT, JSPS KAKENHI Grant Number JP19H03439 to TN, NIH R01 HL049277 to NMS, Developmental Research Program Grant from the Yale Head and Neck SPORE NIDCR P50-DE030707 to ALA, NIH R00 CA225633 to HG. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

Funders	Funder number
Office of Research Infrastructure Programs, National Institutes of Health
National Institutes of Health (NIH)
National Heart, Lung, and Blood Institute (NHLBI)	R01HL049277
National Institute of Dental and Craniofacial Research	P50DE030707
Japan Society for the Promotion of Science	P50-DE030707, R00 CA225633, R01 HL049277, JP19H03439, 23K24098
National Childhood Cancer Registry – National Cancer Institute	R01CA251067, P50CA070907, R00CA225633
National Institute of General Medical Sciences	P20GM121327

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title = "Lysyl hydroxylase 2 mediated collagen post-translational modifications and functional outcomes",

abstract = "Lysyl hydroxylase 2 (LH2) is a member of LH family that catalyzes the hydroxylation of lysine (Lys) residues on collagen, and this particular isozyme has been implicated in various diseases. While its function as a telopeptidyl LH is generally accepted, several fundamental questions remain unanswered: 1. Does LH2 catalyze the hydroxylation of all telopeptidyl Lys residues of collagen? 2. Is LH2 involved in the helical Lys hydroxylation? 3. What are the functional consequences when LH2 is completely absent? To answer these questions, we generated LH2-null MC3T3 cells (LH2KO), and extensively characterized the type I collagen phenotypes in comparison with controls. Cross-link analysis demonstrated that the hydroxylysine-aldehyde (Hylald)-derived cross-links were completely absent from LH2KO collagen with concomitant increases in the Lysald-derived cross-links. Mass spectrometric analysis revealed that, in LH2KO type I collagen, telopeptidyl Lys hydroxylation was completely abolished at all sites while helical Lys hydroxylation was slightly diminished in a site-specific manner. Moreover, di-glycosylated Hyl was diminished at the expense of mono-glycosylated Hyl. LH2KO collagen was highly soluble and digestible, fibril diameters were diminished, and mineralization impaired when compared to controls. Together, these data underscore the critical role of LH2-catalyzed collagen modifications in collagen stability, organization and mineralization in MC3T3 cells.",

author = "Masahiko Terajima and Yuki Taga and Tomoyuki Nakamura and Guo, {Hou Fu} and Yukako Kayashima and Nobuyo Maeda-Smithies and Kshitij Parag-Sharma and Kim, {Jeong Seon} and Amelio, {Antonio L.} and Kazunori Mizuno and Kurie, {Jonathan M.} and Mitsuo Yamauchi",

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doi = "10.1038/s41598-022-18165-0",

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AU - Terajima, Masahiko

AU - Taga, Yuki

AU - Nakamura, Tomoyuki

AU - Guo, Hou Fu

AU - Kayashima, Yukako

AU - Maeda-Smithies, Nobuyo

AU - Parag-Sharma, Kshitij

AU - Kim, Jeong Seon

AU - Amelio, Antonio L.

AU - Mizuno, Kazunori

AU - Kurie, Jonathan M.

AU - Yamauchi, Mitsuo

PY - 2022/12

Y1 - 2022/12

N2 - Lysyl hydroxylase 2 (LH2) is a member of LH family that catalyzes the hydroxylation of lysine (Lys) residues on collagen, and this particular isozyme has been implicated in various diseases. While its function as a telopeptidyl LH is generally accepted, several fundamental questions remain unanswered: 1. Does LH2 catalyze the hydroxylation of all telopeptidyl Lys residues of collagen? 2. Is LH2 involved in the helical Lys hydroxylation? 3. What are the functional consequences when LH2 is completely absent? To answer these questions, we generated LH2-null MC3T3 cells (LH2KO), and extensively characterized the type I collagen phenotypes in comparison with controls. Cross-link analysis demonstrated that the hydroxylysine-aldehyde (Hylald)-derived cross-links were completely absent from LH2KO collagen with concomitant increases in the Lysald-derived cross-links. Mass spectrometric analysis revealed that, in LH2KO type I collagen, telopeptidyl Lys hydroxylation was completely abolished at all sites while helical Lys hydroxylation was slightly diminished in a site-specific manner. Moreover, di-glycosylated Hyl was diminished at the expense of mono-glycosylated Hyl. LH2KO collagen was highly soluble and digestible, fibril diameters were diminished, and mineralization impaired when compared to controls. Together, these data underscore the critical role of LH2-catalyzed collagen modifications in collagen stability, organization and mineralization in MC3T3 cells.

AB - Lysyl hydroxylase 2 (LH2) is a member of LH family that catalyzes the hydroxylation of lysine (Lys) residues on collagen, and this particular isozyme has been implicated in various diseases. While its function as a telopeptidyl LH is generally accepted, several fundamental questions remain unanswered: 1. Does LH2 catalyze the hydroxylation of all telopeptidyl Lys residues of collagen? 2. Is LH2 involved in the helical Lys hydroxylation? 3. What are the functional consequences when LH2 is completely absent? To answer these questions, we generated LH2-null MC3T3 cells (LH2KO), and extensively characterized the type I collagen phenotypes in comparison with controls. Cross-link analysis demonstrated that the hydroxylysine-aldehyde (Hylald)-derived cross-links were completely absent from LH2KO collagen with concomitant increases in the Lysald-derived cross-links. Mass spectrometric analysis revealed that, in LH2KO type I collagen, telopeptidyl Lys hydroxylation was completely abolished at all sites while helical Lys hydroxylation was slightly diminished in a site-specific manner. Moreover, di-glycosylated Hyl was diminished at the expense of mono-glycosylated Hyl. LH2KO collagen was highly soluble and digestible, fibril diameters were diminished, and mineralization impaired when compared to controls. Together, these data underscore the critical role of LH2-catalyzed collagen modifications in collagen stability, organization and mineralization in MC3T3 cells.

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Lysyl hydroxylase 2 mediated collagen post-translational modifications and functional outcomes

Abstract

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