A scalable lysyl hydroxylase 2 expression system and luciferase-based enzymatic activity assay

Hou Fu Guo; Eun Jeong Cho; Ashwini K. Devkota; Yulong Chen; William Russell; George N. Phillips; Mitsuo Yamauchi; Kevin N. Dalby; Jonathan M. Kurie

doi:10.1016/j.abb.2017.02.003

A scalable lysyl hydroxylase 2 expression system and luciferase-based enzymatic activity assay

Hou Fu Guo
, Eun Jeong Cho
, Ashwini K. Devkota
, Yulong Chen
, William Russell
, George N. Phillips
, Mitsuo Yamauchi
, Kevin N. Dalby
, Jonathan M. Kurie

Research output: Contribution to journal › Article › peer-review

13 Scopus citations

Abstract

Hydroxylysine aldehyde-derived collagen cross-links (HLCCs) accumulate in fibrotic tissues and certain types of cancer and are thought to drive the progression of these diseases. HLCC formation is initiated by lysyl hydroxylase 2 (LH2), an Fe(II) and α-ketoglutarate (αKG)-dependent oxygenase that hydroxylates telopeptidyl lysine residues on collagen. Development of LH2 antagonists for the treatment of these diseases will require a reliable source of recombinant LH2 protein and a non-radioactive LH2 enzymatic activity assay that is amenable to high throughput screens of small molecule libraries. However, LH2 protein generated using E coli– or insect-based expression systems is either insoluble or enzymatically unstable, and the LH2 enzymatic activity assays that are currently available measure radioactive CO₂ released from ¹⁴C-labeled αKG during its conversion to succinate. To address these deficiencies, we have developed a scalable process to purify human LH2 protein from Chinese hamster ovary cell-derived conditioned media samples and a luciferase-based assay that quantifies LH2-dependent conversion of αKG to succinate. These methodologies may be applicable to other Fe(II) and αKG-dependent oxygenase systems.

Original language	English
Pages (from-to)	45-51
Number of pages	7
Journal	Archives of Biochemistry and Biophysics
Volume	618
DOIs	https://doi.org/10.1016/j.abb.2017.02.003
State	Published - Mar 15 2017

Bibliographical note

Publisher Copyright:
© 2017 Elsevier Inc.

Funding

This work was supported by NIH grants P50-CA70907-15 (JMK) and R01 CA105155 (JMK, MY), the Welch Foundation (F-1390) (KND), and CPRIT grants RP160652 (JMK, GNP), RP160657 (KND), and RP110532-P1 (KND).

Funders	Funder number
National Institutes of Health (NIH)	P50-CA70907-15, R01 CA105155
National Childhood Cancer Registry – National Cancer Institute	P50CA070907
Welch Foundation	F-1390
Cancer Prevention and Research Institute of Texas	RP160657, RP110532-P1, RP160652

UN SDGs

This output contributes to the following UN Sustainable Development Goals (SDGs)

SDG 3 Good Health and Well-being

Keywords

Chinese hamster ovary cell
Collagen
High-throughput assay
Lysyl hydroxylase 2
Oxygenase
Succinate detection

ASJC Scopus subject areas

Biophysics
Biochemistry
Molecular Biology

Access to Document

10.1016/j.abb.2017.02.003

Cite this

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title = "A scalable lysyl hydroxylase 2 expression system and luciferase-based enzymatic activity assay",

abstract = "Hydroxylysine aldehyde-derived collagen cross-links (HLCCs) accumulate in fibrotic tissues and certain types of cancer and are thought to drive the progression of these diseases. HLCC formation is initiated by lysyl hydroxylase 2 (LH2), an Fe(II) and α-ketoglutarate (αKG)-dependent oxygenase that hydroxylates telopeptidyl lysine residues on collagen. Development of LH2 antagonists for the treatment of these diseases will require a reliable source of recombinant LH2 protein and a non-radioactive LH2 enzymatic activity assay that is amenable to high throughput screens of small molecule libraries. However, LH2 protein generated using E coli– or insect-based expression systems is either insoluble or enzymatically unstable, and the LH2 enzymatic activity assays that are currently available measure radioactive CO2 released from 14C-labeled αKG during its conversion to succinate. To address these deficiencies, we have developed a scalable process to purify human LH2 protein from Chinese hamster ovary cell-derived conditioned media samples and a luciferase-based assay that quantifies LH2-dependent conversion of αKG to succinate. These methodologies may be applicable to other Fe(II) and αKG-dependent oxygenase systems.",

keywords = "Chinese hamster ovary cell, Collagen, High-throughput assay, Lysyl hydroxylase 2, Oxygenase, Succinate detection",

author = "Guo, \{Hou Fu\} and Cho, \{Eun Jeong\} and Devkota, \{Ashwini K.\} and Yulong Chen and William Russell and Phillips, \{George N.\} and Mitsuo Yamauchi and Dalby, \{Kevin N.\} and Kurie, \{Jonathan M.\}",

note = "Publisher Copyright: {\textcopyright} 2017 Elsevier Inc.",

year = "2017",

month = mar,

day = "15",

doi = "10.1016/j.abb.2017.02.003",

language = "English",

volume = "618",

pages = "45--51",

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TY - JOUR

T1 - A scalable lysyl hydroxylase 2 expression system and luciferase-based enzymatic activity assay

AU - Guo, Hou Fu

AU - Cho, Eun Jeong

AU - Devkota, Ashwini K.

AU - Chen, Yulong

AU - Russell, William

AU - Phillips, George N.

AU - Yamauchi, Mitsuo

AU - Dalby, Kevin N.

AU - Kurie, Jonathan M.

PY - 2017/3/15

Y1 - 2017/3/15

N2 - Hydroxylysine aldehyde-derived collagen cross-links (HLCCs) accumulate in fibrotic tissues and certain types of cancer and are thought to drive the progression of these diseases. HLCC formation is initiated by lysyl hydroxylase 2 (LH2), an Fe(II) and α-ketoglutarate (αKG)-dependent oxygenase that hydroxylates telopeptidyl lysine residues on collagen. Development of LH2 antagonists for the treatment of these diseases will require a reliable source of recombinant LH2 protein and a non-radioactive LH2 enzymatic activity assay that is amenable to high throughput screens of small molecule libraries. However, LH2 protein generated using E coli– or insect-based expression systems is either insoluble or enzymatically unstable, and the LH2 enzymatic activity assays that are currently available measure radioactive CO2 released from 14C-labeled αKG during its conversion to succinate. To address these deficiencies, we have developed a scalable process to purify human LH2 protein from Chinese hamster ovary cell-derived conditioned media samples and a luciferase-based assay that quantifies LH2-dependent conversion of αKG to succinate. These methodologies may be applicable to other Fe(II) and αKG-dependent oxygenase systems.

AB - Hydroxylysine aldehyde-derived collagen cross-links (HLCCs) accumulate in fibrotic tissues and certain types of cancer and are thought to drive the progression of these diseases. HLCC formation is initiated by lysyl hydroxylase 2 (LH2), an Fe(II) and α-ketoglutarate (αKG)-dependent oxygenase that hydroxylates telopeptidyl lysine residues on collagen. Development of LH2 antagonists for the treatment of these diseases will require a reliable source of recombinant LH2 protein and a non-radioactive LH2 enzymatic activity assay that is amenable to high throughput screens of small molecule libraries. However, LH2 protein generated using E coli– or insect-based expression systems is either insoluble or enzymatically unstable, and the LH2 enzymatic activity assays that are currently available measure radioactive CO2 released from 14C-labeled αKG during its conversion to succinate. To address these deficiencies, we have developed a scalable process to purify human LH2 protein from Chinese hamster ovary cell-derived conditioned media samples and a luciferase-based assay that quantifies LH2-dependent conversion of αKG to succinate. These methodologies may be applicable to other Fe(II) and αKG-dependent oxygenase systems.

KW - Chinese hamster ovary cell

KW - Collagen

KW - High-throughput assay

KW - Lysyl hydroxylase 2

KW - Oxygenase

KW - Succinate detection

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UR - https://www.scopus.com/pages/publications/85013666741#tab=citedBy

U2 - 10.1016/j.abb.2017.02.003

DO - 10.1016/j.abb.2017.02.003

M3 - Article

C2 - 28216326

AN - SCOPUS:85013666741

SN - 0003-9861

VL - 618

SP - 45

EP - 51

JO - Archives of Biochemistry and Biophysics

JF - Archives of Biochemistry and Biophysics

ER -

A scalable lysyl hydroxylase 2 expression system and luciferase-based enzymatic activity assay

Abstract

Bibliographical note

Funding

UN SDGs

Keywords

ASJC Scopus subject areas

Access to Document

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