Biochemistry of Collagens, Laminins and Elastin: Structure, Function and Biomarkers

By Morten Karsdal

Biochemistry of Collagens, Laminins, and Elastin: Structure, Function and Biomarkers, Third Edition provides current data on key structural proteins (collagens, laminins, and elastin), reviews on how these molecules affect pathologies, and information on how selected modifications of these proteins can result in altered signaling properties of the original extracellular matrix (ECM). Further, it discusses the novel concept that an increasing number of components of the extracellular matrix harbor cryptic signaling functions with ties to endocrine function, and how this knowledge may be used to modulate various pathologies, including fibrotic disease.

This new edition has been expanded and revised to incorporate recent research advances. Several new chapters explore a range of chronic diseases in which the ECM and collagens, laminin and elastin are central players in disease modulation, including new chapters on lung, skin and intestinal disease, as well as cancers. The new edition also considers emerging analytical technologies that can detect biomarkers of ECM degradation, with discussion of protein quantification and detecting aging of collagens.

• Provides an updated, comprehensive discussion of collagen and related structural proteins
• Contains insights into biochemical interactions and changes to structural composition of proteins in disease states
• Proves the importance of proteins for collagen assembly, function and durability
• Examines details on how collagens play a key role in a range of chronic diseases
• Offers approaches for protein quantification and detection of collagen aging

• Language: English
• Publisher: Academic Press
• Release date: Nov 7, 2023
• ISBN: 9780443156182

Morten Karsdal

Morten Karsdal has been CEO of Nordic Bioscience A/S since June 2010. Since 2002, Morten Karsdal has focused his research on the discovery and development of novel biochemical markers. This has resulted in the development of more than 15 ELISA assays for detecting biomarkers that have been used for research in the fields of fibrosis, osteoporosis and osteoarthritis. One of these assays has been 510(k) approved by the Food and Drug Administration (FDA) in the USA.

Book preview

Biochemistry of Collagens, Laminins and Elastin

Structure, Function and Biomarkers

Third Edition

Edited by

Morten A. Karsdal

Nordic Bioscience, Herlev, Denmark

Co-editors

Diana J. Leeming

Kim Henriksen

Anne-Christine Bay-Jensen

Signe Holm Nielsen

Cecilie L. Bager

Detlef Schuppan

Sylvie Ricard-Blum

Thomas R. Cox

Eric S. White

Jeffrey H. Miner

Andrea Heinz

Table of Contents

Cover image

Title page

Copyright

List of contributors

Foreword

Preface

Acknowledgments

List of abbreviations

Introduction

Why are collagens and structural proteins important?

Introduction to the matrix—Interstitial and basement membranes

Overall structure of collagens

Collagen synthesis and other essentials

The origin of collagens—Collagen phylogenetics

Collagen turnover as function of age

Why laminins?

Understanding the epitope of a protein

Why do we need to quantify the ECM?

Conclusion

The collagen history

The diversity of the collagen family: Beyond the 28 collagen types

Molecular and supramolecular assembly of collagens

Collagens: A networking family in various contexts

Chapter 1. Type I collagen

Chapter 2. Type II collagen

Soluble biomarkers of type II collagen

Chapter 3. Type III collagen

Biomarkers of type III collagen

Chapter 4. Type IV collagen

Composition and structure of type IV collagen

Type IV collagen diseases

Matrikines from the NC1 domains

Biomarkers of type IV collagen

Chapter 5. Type V collagen

Autoimmunity

Cardiovasuclar disease

Cancer

Biomarkers of type V collagen

Chapter 6. Type VI collagen

Biomarkers of type VI collagen

Chapter 7. Type VII collagen

Expression and function

The role of COLVII in pathologies

Chapter 8. Type VIII collagen

Type VIII collagen

Biomarkers of type VIII collagen

Chapter 9. Type IX collagen

Soluble biomarkers of type IX collagen

Chapter 10. Type X collagen

Biomarkers of type X collagen

Chapter 11. Type XI collagen

Biomarkers of type XI collagen

Chapter 12. Type XII collagen

Chapter 13. Type XIII collagen

Biomarkers of type XIII collagen

Chapter 14. Type XIV collagen

Chapter 15. Type XV collagen

Chapter 16. Type XVI collagen

Role in aging and disease

Biomarkers of type XVI collagen

Chapter 17. Type XVII collagen

Chapter 18. Type XVIII collagen

Text

Tissue expression

Functional role of type XVIII collagen in pathological conditions

Hemophilia and the involvement of the short isoform of type XVIII collagen

The short isoform of type XVIII collagen, a biomarker for hemophilia

Role of endostatin in biology and pathology

Biomarkers of endostatin

Chapter 19. Type XIX collagen

Biomarkers of type XIX collagen

Chapter 20. Type XX collagen

Chapter 21. Type XXI collagen

Chapter 22. Type XXII collagen

Biomarkers of type XXII collagen

Chapter 23. Type XXIII collagen

Biomarkers of type XXIII collagen

Chapter 24. Type XXIV collagen

Chapter 25. Type XXV collagen

Chapter 26. Type XXVI collagen

Chapter 27. Type XXVII collagen

Chapter 28. Type XXVIII collagen

Biomarkers of type XXVIII collagen

Chapter 29. Laminins

Summary

Structure

Nomenclature

Interaction partners

Signaling

Laminin-111

Laminin-211

Laminin-121

Laminin-221

Laminin-332, -311, and -321

Laminin-411

Laminin-421

Laminin-511

Laminin-521

Laminin-213

Laminin-323, -423, and -523

Laminin mutations and disease in humans

Chapter 30. Elastin

Biomarkers of elastin

Chapter 31. Fibronectin

Fibronectin

Fibronectin as a biomarker

Chapter 32. The extended family of collagens

Introduction

Chapter 33. Collagen receptors

Introduction

Integrins

DDRs

The immunoglobulin (Ig) receptor superfamily

CD44

Summary

Chapter 34. Aging—the most important collagen neoepitope?

Introduction

PTMs related to aging

Neoepitope identification

Chapter 35. Collagen chaperones

Chaperone introduction

Chaperone activities of prolyl hydroxylases and peptidyl-prolyl isomerases

HSP47

HSP47 in disease and as a target for treatment

SPARC

Periostin

COMP

Chapter 36. Collagen diseases

Osteogenesis imperfecta

Ehlers–Danlos syndrome

Alport syndrome

Epidermolysis bullosa

Stickler syndrome

Collagen VI-related muscular dystrophies

Chapter 37. The signals of the extracellular matrix

Introduction

Type IV collagen—arresten, canstatin, tumstatin, tetrastatin, pentastatin, and hexastatin

Type VIII collagen—vastatin

Collagen XV—restin

Type XVIII collagen—endostatin

Other extracellular matrix fragments

Chapter 38. The roles of collagens and fibroblasts in cancer

Tumor fibrosis and cancer-associated fibroblasts

Collagens and immunotherapy

Altered degradation of collagen in the tumor

Collagen fragments as noninvasive biomarkers of tumor fibrosis

Chapter 39. Use of extracellular matrix biomarkers in clinical research

Biomarker terminology

Extracellular matrix biomarkers as a translational tool in drug development

Established use of ECM biomarkers in drug development and epidemiology

Perspectives for collagen biomarkers in clinical research

Chapter 40. Common confounders when evaluating noninvasive protein biomarkers

Technical and analytical validation of novel noninvasive protein biomarkers

Laboratory automation

Clinical validation of novel noninvasive biomarkers

Analyte features and impact on pathological/clinical relevance of noninvasive biomarker measurements

Sample handling when evaluating novel noninvasive biomarkers

Importance of patient cohort characteristics, sample size, and reporting guidelines

Chapter 41. Extracellular matrix and endotypes

The extracellular matrix

Endotypes of the ECM

Clinical application of endotyping

Clustering of biochemical markers as indications of endotypes

PRO-C2 as a biomarker of the low cartilage repair endotype in osteoarthritis patients

Chapter 42. The extracellular matrix of the skin: systemic diseases with local manifestations

Introduction

Chapter 43. The basement membrane and its role in pulmonary disease

Assembly, composition and function

Localization, remodeling and repair

Pathological changes in basement membranes

Chapter 44. Collagen remodeling in inflammatory bowel disease

Collagen remodeling and intestinal fibrosis

Biomarkers of intestinal fibrosis and mucosal damage

Antifibrotic treatments for IBD

Chapter 45. ECM biomarkers in population-based cohorts

Key features of population-based studies

Endotrophin, a marker of disease severity

Genome-wide association studies

The importance of a balanced ECM

Chapter 46. Collagen biomarkers of chronic diseases

Index

Copyright

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List of contributors

Marta Alexdottir,     Nordic Bioscience, Herlev, Denmark

A. Arvanitidis,     Biomarkers and Research, Nordic Bioscience, Herlev, Denmark

Cecilie Liv Bager,     Nordic Bioscience, Herlev, Denmark

A.C. Bay-Jensen,     Nordic Bioscience, Herlev, Denmark

Asger R. Bihlet,     NBCD A/S/Sanos Group, Clinical Development, Søborg, Denmark

Helene W. Breisnes,     Nordic Bioscience, Herlev, Denmark

Thomas R. Cox

Matrix & Metastasis Lab, The Garvan Institute of Medical Research & the Kinghorn Cancer Centre, Cancer Ecosystems Program, Sydney, NSW, Australia

School of Clinical Medicine, St Vincent's Healthcare Clinical Campus, Faculty of Medicine and Health, UNSW Sydney, Sydney, NSW, Australia

M. Crespo-Bravo

Department of Biomedical Sciences, University of Copenhagen, Copenhagen, Denmark

Nordic Bioscience, Herlev, Denmark

A. Engstroem,     Nordic Bioscience, Herlev, Denmark

Federica Genovese,     Nordic Bioscience, Herlev, Denmark

F.S. Gillesberg,     Nordic Bioscience, Herlev, Denmark

S.S. Groen

Nordic Bioscience, Herlev, Denmark

Department of Biomedical Sciences, University of Copenhagen, Copenhagen, Denmark

N.S. Gudmann,     Nordic Bioscience, Herlev, Denmark

A.E.M Guiliani,     Biomarkers and Research, Nordic Bioscience, Herlev, Denmark

M.T. Hannani,     Nordic Bioscience, Herlev, Denmark

Annika H. Hansen,     Biomarkers and Research, Nordic Bioscience, Herlev, Denmark

Niels U.B. Hansen,     Nordic Bioscience, Herlev, Denmark

Y. He,     Nordic Bioscience, Herlev, Denmark

A. Heinz,     LEO Foundation Center for Cutaneous Drug Delivery, Department of Pharmacy, University of Copenhagen, Copenhagen, Denmark

Kim Henriksen,     Nordic Bioscience, Herlev, Denmark

S. Holm Nielsen

Department of Biotechnology and Biomedicine, Technical University of Denmark, Lyngby, Denmark

Nordic Bioscience, Herlev, Denmark

S.M. Jansen,     Nordic Bioscience, Herlev, Denmark

C. Jensen,     Nordic Bioscience, Herlev, Denmark

H. Jessen,     Nordic Bioscience, Herlev, Denmark

Pernille Juhl,     Nordic Bioscience, Herlev, Denmark

M.A. Karsdal,     Nordic Bioscience, Herlev, Denmark

S.N. Kehlet,     Nordic Bioscience, Herlev, Denmark

N.G. Kjeld,     Nordic Bioscience, Herlev, Denmark

J.H. Kristensen,     Nordic Bioscience, Herlev, Denmark

L.L. Langholm,     Nordic Bioscience, Herlev, Denmark

Clara F.G. Laursen,     Nordic Bioscience, Herlev, Denmark

D.J. Leeming,     Nordic Bioscience, Herlev, Denmark

M. Lindholm,     Nordic Bioscience, Herlev, Denmark

I. Lønsmann,     Nordic Bioscience, Herlev, Denmark

Y.Y. Luo,     Nordic Bioscience, Herlev, Denmark

E.A. Madsen,     Nordic Bioscience, Herlev, Denmark

S.F. Madsen

Nordic Bioscience, Herlev, Denmark

University of Copenhagen, Copenhagen, Denmark

T. Manon-Jensen,     Biomarkers and Research, Nordic Bioscience, Herlev, Denmark

Jeffrey H. Miner,     Division of Nephrology, Washington University School of Medicine, St. Louis, MO, United States

J.H. Mortensen,     Nordic Bioscience, Herlev, Denmark

A.L. Møller,     Nordic Bioscience, Herlev, Denmark

M.J. Nielsen,     Nordic Bioscience, Herlev, Denmark

Neel I. Nissen,     Nordic Bioscience, Herlev, Denmark

R.S. Pedersen,     Department of Biomedical Sciences, University of Copenhagen, Copenhagen, Denmark

M. Pehrsson,     Nordic Bioscience, Herlev, Denmark

H. Port

Nordic Bioscience, Herlev, Denmark

Department of Biomedical Sciences, University of Copenhagen, Copenhagen, Denmark

Daniel G.K. Rasmussen,     Nordic Bioscience, Herlev, Denmark

A.L. Reese-Petersen,     Nordic Bioscience, Herlev, Denmark

Sylvie Ricard-Blum,     Institut de Chimie et Biochimie Moléculaires et Supramoléculaires (ICBMS), UMR 5246, University Claude Bernard Lyon 1, Villeurbanne Cedex, France

S.R. Rønnow,     Nordic Bioscience, Herlev, Denmark

Jannie M.B. Sand,     Nordic Bioscience, Herlev, Denmark

S. Sardar,     Nordic Bioscience, Herlev, Denmark

A.S. Siebuhr,     Nordic Bioscience, Herlev, Denmark

D. Sinkeviciute,     Nordic Bioscience, Herlev, Denmark

N. Sparding,     Nordic Bioscience, Herlev, Denmark

S. Sun,     Nordic Bioscience, Herlev, Denmark

P.M. Szlarski,     Nordic Bioscience, Herlev, Denmark

J. Thorlacius-Ussing,     Nordic Bioscience, Herlev, Denmark

Christian S. Thudium,     Nordic Bioscience, Herlev, Denmark

I.F. Villesen,     Nordic Bioscience, Herlev, Denmark

Eric S. White,     Boehringer Ingelheim Pharmaceuticals, Inc., Ridgefield, CT, United States

Nicholas Willumsen,     Nordic Bioscience, Herlev, Denmark

Foreword

Biochemistry of Collagens, Laminins and Elastin by Morten A. Karsdal and colleagues has started as a comprehensive edition on collagens and major noncollagen extracellular matrix proteins 4 years ago. Meanwhile, this multiauthored book, all by prime experts in their field, has become a unique resource that focuses mainly, but not exclusively on 28 collagen types that are all trimeric molecules composed of identical or homologous collagen alpha chains. Their key structural characteristic is that they contain long or short sequences of collagen chain-specific repetitive amino acid triplets, often Gly-Pro-Hyp, that permit the formation of collagen triple helices, which lend structural stability and allow for formation of large supramolecular structures. While the fibril-forming collagens like types I, II, III, and V form the typical collagen fibrils by lateral alignment, basement membrane collagen type IV and the microfilamentous collagen type VI form networks and interfibrillar flexible cables, respectively. This was known established during the early days of collagen biochemistry 4–5 decades ago, using collagen chain N-terminal sequence analysis of overlapping proteolytic peptides and visualization of supramolecular assemblies by electron microscopical technologies like rotary shadowing. This went along with the characterization of noncollagenous ECM proteins like fibronectin, laminin, nidogen, and tenascin-C, and the identification of the role of the ECM proteins not only as superstructures that compartmentalize and keep tissues together, but that have central roles in signaling to cells in development, proliferation, differentiation, and dedifferentiation as in cancer and metastasis. Since then, the field has made further dramatic progress, largely by employing DNA/RNA technology, modern protein sequencing and mass spectrometry, and especially in vitro and in vivo models of health and disease. While the key role of the ECM has been demonstrated and recognized in essential organs, and in all processes of cell physiology, the community of biochemists and cell biologists who comprehensively understand the biochemistry, structure, metabolism, and signaling of collagens and major noncollagenous ECM proteins is small.

In this book, however, the group of ECM and collagen experts that combine the protein and genetic information with functional studies and clinical translation is impressive. As in the first edition, there is a superb illustration of the collagens and select noncollagen proteins and their assembly, including an updated focus on functional and translational aspects of these molecules. Thus, on top of or overlapping with the previous 37 chapters, 9 novel chapters have been added that include the collagen history, fibronectins, collagen receptors, new collagen families, ageing and collagen neoepitopes, cancer-associated fibroblasts and the relation to cancer and fibrosis, confounders in evaluation protein biomarkers, ECM and endotypes, skin diseases, basement membranes and related diseases, inflammatory bowel diseases, ECM markers in population based studies, collagen biomarkers in chronic diseases, and collagen neoepitopes to assess the balance of ECM formation and degradation. Most of the functional characteristics of these ECM proteins are only partly predictable by genetic sequencing, such as naturally generated proteolytic fragments with unique signaling properties modulating not only cellular differentiation, proliferation, and migration, but also angiogenesis and metabolism. These properties of certain collagens, other matrix components, and their fragments are already being exploited for novel therapies to address organ fibrosis, inflammation, and cancer. Moreover, the utility of serum markers that derive from specific matrix components, especially the collagen neoepitope makers that derive from their defined naturally produced proteolytic fragments, are outlined in detail. This is a rapidly evolving field that permits a noninvasive assessment of the dynamics of matrix synthesis or degradation in an organ- and disease-specific way. The generation of reproducible ELISAs for direct, i.e., ECM-derived, serum markers of disease and of altered collagen and ECM turnover has already yielded key predictive and activity markers for fibrosis, vascular or metabolic disease, and cancer. These biomarkers are beginning to revolutionize case finding in population studies and permit early monitoring of drug development, e.g., in clinical studies for liver, lung, cardiovascular, and kidney fibrosis or cancer that otherwise have to be based on large cohorts subjected to sequential tissue biopsies, or difficult-to-interpret functional readouts.

As already 4 years ago, and even more now with the third edition, there is no single review nor assembly of reviews that covers all these areas of collagen and ECM research and its translation in a comparably comprehensive way and across narrow professional boundaries, as this book does. I consider it the best available overview and resource that will be an invaluable asset for every researcher, clinician, and drug developer with an interest in ECM biology, biochemistry, and translational perspectives.

Detlef Schuppan, MD, Ph.D.

Professor of Medicine

Mainz University Medical Center and Harvard Medical School

Preface

This book on extracellular matrix proteins is a result of a love of matrix biology, structural proteins, and the effects they have on cellular function and fate. These amazing proteins control the life, death, and fate of cells. This book has also been created as a result of the fascinating discovery that many proteins, when quantified with technologies targeting different domains, reveal very specific and important clues to biology and disease progression. We need to better understand the ECM and the protein-specific subdomain epitopes, together with their precise and accurate quantification, to advance science for the benefit of patients.

The physiological and pathophysiological roles and functions of many collagens remain to be discovered. We hope this book will inspire new researchers to take on the collagen challenge and present novel research and biology that is crucial for understanding the extracellular matrix in pathological and physiological conditions. Importantly, the collagens are intriguingly diverse, albeit they share many similarities.

Today, the medical field is facing the challenge of improved personalized medicine. The base substance of all organs is ECM proteins, mostly collagens. Consequently, most chronic diseases involve changes in these essential proteins. We need to correctly quantify the right epitope of the protein, not just the total protein, to change the lives of patients.

This book aims to (1) summarize the available data of key structural proteins of the extracellular matrix (i.e., collagens, laminins, and elastin) and their effect on cells; (2) review how these molecules affect pathologies, as exemplified by monogenetic disorders; (3) describe the selected posttranslational modifications (PTMs) of ECM proteins that result in altered collagen signaling properties of the original ECM component, showing these collagens harbor cryptic signaling functions that may be viewed as endocrine functions; (4) focus on the common confounders for serological measurements that we need to understand to better quantify tissue turnover; (5) discuss the important collagen-binding and modulating proteins, the collagen chaperones, and the collagen machinery; and (6) discuss the use of collagen biomarkers in clinical research, along with the need for quantifying tissue formation and degradation separately.

With this book, we hope to inspire more research into ECM proteins, particularly collagens, in the quest to understand the right epitope of the protein. This will facilitate the understanding of pathologies and assist in developing improved diagnostic tools to help patients.

Sincerely,

M.A. Karsdal, MSc, Ph.D., mMBA,

Professor, SDU

Acknowledgments

I wish to thank Claus Christiansen for discovering, developing, and FDA-validating the first biomarker of ECM remodeling, CTX-I, a fantastic collagen degradation fragment made by cathepsin K of type I collagen, which is now recognized as the standard bone resorption marker. This discovery has inspired many researchers, including myself, to uncover, develop, and validate the biomarkers of the extracellular matrix. Claus has always inspired us to do crazy, impossible, but focused science, with the end goal of forwarding science by providing research that applies to many other fields and researchers.

I want to thank all the present and former Ph.D. students, as well as the senior researchers, who have helped me understand and quantify the ECM. Without your dedication and hard work in generating assays and data, this book would have been impossible. Further, special thanks to all the excellent technicians who helped in generating novel and impossible assays of the matrix and measuring essential samples correctly.

This book is a team effort of a large group of ECM researchers dedicated to quantifying and trying to understand the matrix in both pathological and physiological conditions. Thank you for all your help with the book.

Most importantly, I wish to thank all the former, current, and future collaborators who provided samples and discussions that help us understand the role of ECM in connective tissue biology.

Finally, I wish to thank the Danish Research Foundation for making it possible to write this book by supporting Ph.D. programs, research on ECM, biomarkers, and excellence in science.

Sincerely,

M.A. Karsdal

List of abbreviations

97-LAD    97 kD linear IgA dermatosis antigen

aa    Amino acid

ADAM    A disintegrin and metalloproteinase

Alpha1    Alpha 1 chain

Alpha2    Alpha 2 chain

ANCAs    Antineutrophil cytoplasmic antibodies

APP    Amyloid precursor protein

ASPD    Antisocial personality disorder

BACE1    β-site APP-cleaving enzyme 1

bFGF    Basic fibroblast growth factor

BM    Bethlem myopathy

BMP-1    Bone morphogenetic protein 1

BMZ    Basement membrane zone

BP180    180kD bullous pemphigoid antigen

BP230    230-kD bullous pemphigoid antigen

C5M    Matrix metalloproteinase fragment of type V collagen

CCDD    Congenital cranial dysinnervation disorder

CIA    Collagen-induced arthritis

CLAC    Collagen-like amyloidogenic component

COL    Collagenous domain

COPD    Chronic obstructive pulmonary disease

DDR1    Discoidin domain receptor 1

DMD    Duchenne muscular dystrophy

ECM    Extracellular matrix

EDS    Ehlers-Danlos syndrome

eGFR    Estimated glomerular filtration rate

ELISA    Enzyme-linked immunosorbent assay

EMI    Emilin

EMID2    Emilin/multimerin domain-containing protein 2

FACIT    Fibril-associated collagens with interrupted triple helices

FN    Fibronectin type III

G    Globular

GBM    Glomerular basement membrane

HANAC    Hereditary angiopathy with nephropathy, aneurysms, and muscle cramps

HGNC    HUGO Gene Nomenclature Committee

HNE    Human neutrophil elastase

HNSCC    Squamous cell carcinoma of the head and neck

HPLC-MS    High-performance liquid chromatography–mass spectrometry

HSGAG    Heparan sulfate glycosaminoglycan

IGFBP-5    Insulin-like growth factor binding protein-5

IHC    Immunohistochemistry

IPF    Idiopathic pulmonary fibrosis

ISEMFs    Intestinal subepithelial myofibroblasts

JEB    Junctional epidermolysis bullosa

KO    Knockout

LAD-1    120 kD linear IgA dermatosis antigen

LG    Laminin globular

MI    Myocardial infarction

MIM    Mendelian inheritance in man

MMPs    Matrix metalloproteinases

mRNA    Messenger ribonucleic acid

MTJs    Myotendinous junctions

NAG    N-acetyl-β-D-glucosaminidase

NC1    Noncollagenous 1

NF    Nuclear factor

NSCLC    Nonsmall-cell lung carcinoma

OA    Osteoarthritis

OSCC    Oral squamous cell carcinoma

P5CP    C-terminal propeptide of type V collagen

P5NP    N-terminal propeptide of type V collagen

PARP    Proline-arginine-rich protein

PDGF    Platelet-derived growth factor

Pro-C5    Neoepitope of the C-terminal propeptide of type V collagen

SNP    Single nucleotide polymorphism

SVAS    Supravalvular aortic stenosis

TACE    TNF-α converting enzyme

TCR    T-cell receptor

Tgase    Transglutaminase

TGF-β    Transforming growth factor-β

TSP    Thrombospondin

TSPN    Thrombospondin N-terminal-like domain

TSPN-1    N-terminal domain of thrombospondin-1

UCMD    Ullrich congenital muscular dystrophy

vWF-A    Type A domains of von Willebrand factor

Introduction

M.A. Karsdal

Nordic Bioscience, Herlev, Denmark

Abstract

In bone, skin, and connective tissues, type I collagen is not only the most abundant collagen, but it is also essential for tissue integrity. Type I collagen is an interstitial matrix molecule that is highly organized in fibrils. Its importance is illustrated by osteogenesis imperfecta and Ehlers–Danlos syndrome, which are severe diseases caused by mutations in the genes for COL1A1 and COL1A2. Type I collagen is heavily modified post-translationally, with a series of modifications that occur during synthesis, including interhelical and interfibrillar cross-links; this ensures the integrity of the helix. Further, it is also post-translationally modified during regular aging and disease, and these modifications include cleavage, isomerization, and glycation, which, in some cases, contribute to the deterioration of the integrity of the matrix. The post-translational modifications of collagen type I have proven highly useful as biomarkers of synthesis and degradation of the matrix, especially in bone diseases, where the discovery of the cathepsin K–generated degradation fragment CTX has completely changed the field. Other markers, such as the N- and C-terminal propeptides, have also been used extensively to monitor the synthesis of the bone matrix. Finally, more recently, matrix metalloproteinase (MMP)-generated fragments of type I collagen have shown a strong relation to systemic inflammation and can be used to monitor the efficacy of anti-inflammatory therapies.

Keywords: Biomarkers, Bone turnover, Collagen type I, Therapy

The backbone of tissues is composed of structural proteins, such as collagens, elastin, and laminins. In fact, type I collagen is the most abundant protein in the body, and there are eight collagens on the top 20 list of the most abundant proteins [1]. A central concept that often is overlooked in extracellular matrix (ECM) and tissue research is that the body regenerates. The bones regenerate every 10–25 years [2]; the liver can regenerate from only a quarter complete, and the epithelium of the intestine regenerates every week [3]. Consequently, formation and degradation processes are ongoing. Aligning with this, when we quantify biomarkers, we need to translate those biomarkers back to a consequence of either their formation or degradation processes, rather than a correlation to these [4]. To interpret pharmacodynamic biomarker changes, we need to know if we inhibit the degradation of tissues or are stimulating formation. In fact, this concept has changed the bone field, resulting in treatments for osteoporosis and low bone mass that either inhibit bone resorption or stimulate bone formation [5]. This was a result of the availability of very accurate and specific biomarkers of both bone formation or bone degradation [6,7]. This can be achieved in many other chronic diseases by understanding tissue balance. But research is needed within basic and applied science to refine biomarkers.

During tissue turnover, proteins are formed and degraded in a tight equilibrium to ensure tissue health and homeostasis. Imbalances in these processes can result in tissue accumulation (i.e., fibrosis) [8]. Fibrosis can affect almost any organ or tissue and is considered a pathological accumulation of tissue in which the newly formed tissue has a different protein composition [9]. In direct alignment with the former paragraph, fibrosis may arise from either an excessive tissue formation or impaired degradation of tissues [10], which most likely should be treated differently [10]. Equally important, though, the proteins are also orientated differently, in pathologies versus healthy tissue, such as woven bone in high bone turnover diseases and fibrosis [11]. Thus, fibrosis is both a protein accumulation and disorientation/mal-localization pathology [9]. In fact, a good protein (collagen) may be a bad collagen in different places [9,12,13]; consequently, the exact function of the individual collagens becomes very important.

Collagens are not just collagens; each collagen has unique expression patterns. Some collagens not only have simple structural functions, but they are also key for signaling [14]. The common denominator for collagens is the triple helix structure, which is less pronounced in laminins. Collagens are divided into several distinct subgroups, of which the fibrillar and networking collagens are the most investigated. This chapter introduces the superstructure of collagens, elastin, and laminins, as well as the essentials of collagen biology, expression, and function. Furthermore, this chapter highlights the need for quantifying ECM turnover, which is essential for the progression of many pathologies and that only can be accomplished by appropriate biomarkers [1,15].

Why are collagens and structural proteins important?

Pathological tissue turnover can affect almost any organ or tissue, albeit in different ways [16,17]. Fig. 1 illustrates the major fibroproliferative and tissue destruction diseases that have a significant impact on human health, which is more than 50 in total, and many chronic illnesses with a fatal outcome. Fibrosis is characterized by the formation of excess connective tissue that damages the structure and function of the underlying organ or tissue and can lead to a wide variety of diseases [18,19].

Figure 1  Examples of fibroproliferative diseases in different organs. AMD, age-related macular degeneration; COPD, chronic obstructive pulmonary disease; IPF, idiopathic pulmonary fibrosis; NASH, nonalcoholic steatohepatitis; PAH, pulmonary arterial hypertension [8,14].

Forty-five percent of all deaths in the developed world are associated with chronic fibroproliferative diseases [16], such as atherosclerosis and alcoholic liver disease. The common denominator of fibroproliferative diseases is increased fibroblast activity, leading to dysregulated tissue remodeling. Remodeling can eventually result in the excessive and abnormal accumulation of ECM components in the affected tissues and, ultimately, in tissue failure [20]. This ECM has an altered structure and signals abnormally to the cells that are embedded in it [13,21]. The ECM composition changes dramatically during the development of fibrosis because the proteins interact with each other and the cells that attach to them [3,9].

For a long time, fibrotic tissue was considered an inactive scaffold, preventing the regeneration of the affected organ. However, this perception still does not hold true because fibrosis is neither static nor irreversible, but instead, it is the result of a continuous remodeling process and, hence, is susceptible to intervention [16,17,22,23]. The future challenge in fibrosis will be to halt fibrogenesis and reverse advanced fibrosis without affecting tissue homeostasis or interfering with normal wound healing [24–27]. Consequently, our increased understanding of the ECM, its dynamics, and the potential of fibrotic microenvironments to reverse holds promise for the development of highly specific antifibrotic therapies with minimal side effects. With emerging tools enabling assessment of both formation and degradation of the ECM separately [10], the understanding of the tissue balance may be further investigated and used for the benefit of patients.

Traditionally, only growth factors, cytokines, hormones, and other small molecules have been considered relevant mediators of inter-, para-, and intracellular communication and signaling. However, the ECM fulfills direct and indirect paracrine—or even endocrine—roles [28]. In addition to maintaining the structure of tissues, the ECM has properties that directly signal to cells. Even conceptually, exclusive structural proteins such as fibrillar collagens or proteoglycans are emerging as specific signaling molecules that affect cell behavior and phenotype via cellular ECM receptors [9]. In addition, the ECM can bind to otherwise soluble proteins, growth factors, cytokines, chemokines, or enzymes, restricting or regulating their access to cells, as well as specifically attracting and modulating the cells producing these factors. Moreover, specific proteolysis can generate biologically active fragments from the ECM, while the parent molecules of the ECM are inactive. One such fragment is endostatin, a famous fragment of type XVIII collagen [29]. This and other collagen fragments will be discussed in a later chapter. Thus, the ECM can control cell phenotype by functioning as a precursor bank of potent signaling fragments, in addition to having a direct effect on cell phenotype through ECM cell interactions mediated by receptors such as integrins and/or certain proteoglycans [30–32].

Introduction to the matrix—Interstitial and basement membranes

There are two main ECM areas in the body: the basement membrane and interstitial matrix. The basement membrane is the underlying endothelium and epithelium holding these cells in a permeable loose matrix and ensuring that the cells are polarized and functionable. The main constituents of the basement membrane are type IV collagen, nidogen, and laminins [30,33–36]. The interstitial matrix is responsible for the structure and rigidity of tissues and is mainly produced by fibroblasts [37]. The primary proteins in the interstitial matrix are collagens, which are mostly produced by fibroblasts. The main collagens of the interstitial matrix are type I, III, V, and VI [38]. When a tissue is injured, endothelial or epithelial cells on the tissue surface are destroyed, exposing the basement membrane to degradation and an influx of inflammatory cells and the deeper interstitial membrane to the risk of fibrosis, Fig. 2. This tissue turnover results in the release of protein fragments, of which some are potent signaling fragments, as will be discussed later.

Figure 2  Schematic representation of the generation from the basement membrane and interstitial membrane of the biomarkers of endothelial or epithelial cell damage. (A) Overview of the endothelial cell layer which has a well-organized basement and interstitial membranes below. (B) Cell damage results in the death of localized epithelial cells, resulting in the creation of myofibroblasts from fibroblasts which proliferate and form a matrix (B1), followed by (B2) recruitment of inflammatory cells such as macrophages through the damaged epithelium. (C) Continuous cell insult results in tissue damage and denudation of the epithelium exposing the basement membrane to degradation in which fragments of the basement membrane are released. (D) Deeper tissue damage exposes the underlying interstitial membrane to degradation, and its fragments are released through the basement membrane (D1). Continuous inflammation load and activation of fibroblasts results in overproduction of components of the interstitial and basement membranes in an unorganized manner, i.e., in fibrosis (D2).

Overall structure of collagens

Collagens are widely expressed throughout all organs and tissues; they are the most abundant proteins in connective tissue. There are eight collagens among the 20 most abundant proteins in the body. Type I collagen is the most abundant protein [31,39]. The eight collagens on the list of the most abundant proteins are collagen types I, II, III, IV, V, VI, IX, and IX. To date, 46 different collagen genes coding for 28 different types of collagens have been identified [40]. Fig. 3 schematically displays the primary structure of the molecules. Collagens are trimeric molecules composed of three polypeptide α-chains that contain the repeated sequence (G–X–Y)n, with X being frequently proline and Y hydroxyproline. These repeats allow for the formation of a triple helix, which is the characteristic structural feature of the collagen superfamily. Each member of the collagen family contains at least one triple-helical domain (COL) that is secreted and deposited into the ECM. Most collagens can form supramolecular aggregates. Besides triple-helical domains, collagens contain non-triple-helical (NC) domains, which are used as building blocks by other ECM proteins. The molecular structure and supramolecular assembly of collagens allow for their division into major subfamilies, depending on the supramolecular structure, as depicted in Fig. 4.

1. Fibril-forming collagens (I, II, III, V, XI, XXIV, and XXVII).

2. Fibril-associated collagens with interrupted triple helices (FACITs) (IX, XII, XIV, XVI, XIX, XX, XXI, and XXII). The FACITs do not form fibrils by themselves but are associated with the surface of collagen fibrils.

3. Network-forming collagens (IV, VIII, and X) form a pattern in which four molecules assemble via their amino-terminal 7S domain to form tetramers, while two molecules assemble via their carboxy-terminal NC1 domain to form NC1 dimers.

4. Membrane collagens (XIII, XVII, XXIII, and XXV).

In continuation of the last two paragraphs, the distribution of collagens is schematically illustrated in Fig. 5. The type and location of the collagens are controlled and important in the tissue. Under the epithelial cells, the networking collagens are found, with potential signals, in contrast to the fibrillar and FACIT collagens, which are produced by fibroblasts. The figure combines the overall structure of the individual collagens, highlighting the diversity of each collagen. The position in the ECM and the structure of the different family members are very different, highlighting that the exact type of collagen and the position in the ECM is as important as the total collagen amount [41]. In addition, additional fragments are generated from the collagens by proteolysis during tissue remodeling that have been shown to have potential signaling function are depicted, such as arresten, canstatin, tumstatin, tetrastatin, penstatin, hexastatin, vastatin, restin, endostatin, and endotrophin, which will be discussed intensively in later chapters.

Collagen synthesis and other essentials

The fibrillar collagens, such as type I collagens, are synthesized in the endoplasmic reticulum, where two pro-α1 chains and one pro-α2 combine to form procollagen, as illustrated schematically in Fig. 6. This complex process is mediated by the hydroxylation of prolines and lysines to stabilize the helix, secretion to the extracellular space, enzymatic removal of the N- and C-terminal propeptides, packaging the material into fibrils, and, finally, the formation of intermolecular cross-links that lead to the final and mechanically competent collagen fibrils. Further details on the molecular aspects of this process are outside the scope of the present chapter, and we refer the reader to [42–45] and the specific chapter dedicated to the collagen machinery. Extensive research has been conducted in the biosynthesis of fibril-forming collagens that are synthesized as procollagen molecules comprised of an amino-terminal propeptide followed by a short, nonhelical, N-telopeptide, a central triple helix, a C-telopeptide, and a carboxy-terminal propeptide. Individual procollagen chains are subjected to numerous post-translational modifications (PTMs). In the synthesis process, heat-shock protein 47 (HSP47) binds to procollagen in the endoplasmic reticulum. HSP47 is a specific molecular chaperone of procollagen [46]. The stabilization of the procollagen triple helix at body temperature requires the binding of more than 20 HSP47 molecules per triple helix [36]. It has been suggested recently that intracellular secreted protein acidic and rich in cysteine (SPARC) might be a collagen chaperone because it binds to the triple-helical domain of procollagens and its absence leads to defects in collagen deposition in tissues [47]. In direct alignment, the cleavage of SPARC has been shown to increase collagen affinity and protect against MMP degradation of fibrillar collagens [48]. Both propeptides of the N- and C-terminals of the procollagens are cleaved during the maturation process. The N- and C-terminal propeptides are cleaved proteinases belonging to the A disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS) family and the bone morphogenetic protein-1 (BMP-1) [49–51]. BMP-1 cleaves the carboxy-terminal propeptide of procollagens, except for the carboxy-terminal propeptide of the pro-a1(V) chain, which is processed by furin. This process, in which the actual proteolytic process is releasing the propeptides from the mature intact collagen triple helix and allows them to be incorporated into the matrix structure, is itself subject to regulation. The procollagen C-proteinases (PCPE) can increase BMP-1/tolloid proteinase activity on the c-terminal telopeptide, which results in a remarkable increase in the rate of fibril cleavage by more than fivefold [43]. Finally, telopeptides contain the sites where cross-linking occurs. This process is initiated by the oxidative deamination of lysyl and hydroxylsyl residues catalyzed by the enzymes of the lysyl oxidase family. Several mutations in the collagen synthesis machinery have been linked to different pathologies, as outlined in Table 1, which will be discussed in the collagen machinery chapter. It is important to understand the synthesis of collagens, for researchers to be able to control the synthesis of collagens.

Figure 3  Schematic primary structure of collagens including a depiction of functional domains.

Figure 4  The supramolecular structure of collagens.

Figure 5  Simplified schematic representation of the basement membrane ECM underlying the epithelium, and the interstitial matrix produced by the fibroblasts. The overall structure of the individual collagens is depicted, highlighting the diversity of each collagen despite all being one protein family with a common core structure. It is very clear from the figure that the position in the ECM and the structure of the different family members are very different. In addition, additional fragments are generated from the collagens by proteolysis during tissue remodeling that have been shown to have potential signaling function are depicted, such as arresten, canstatin, tumstatin, tetrastatin, penstatin, hexastatin, vastatin, restin, endostatin and endotrophin. Strongly modified and amended from Refs. [3,12,45].

Figure 6  Biosynthesis of collagen. The process by which type I collagen is made in the endoplasmatic reticulum (ER), folded, processed, and eventually is made into the collagen fibrils, making up the collagen fibers. The top part of the Figure (above the cell membrane) illustrates the intracellular events and the bottom part of the Figure (below the cell membrane) represents the extracellular events. During the synthesis of pro-α chains in the endoplasmatic reticulum, specific peptidyl lysine residues are hydroxylated to form hydroxylysine (–OH–NH2) and, subsequently, specific glycosylated hydroxylysine residues, this latter step being called O-linked glycosylation. For the latter, either single galactose (a red hexagon) or glucose-galactose (two red hexagons) are attached. After these and other modifications (for example, hydroxylation of proline, or asparagine-linked glycosylation shown as closed circles in the C-propeptide), two pro-α1 chains (solid line) and one pro-α2 chain (dotted line) associate with one another and fold into a triple helical molecule from the C- to the N-terminus to form a procollagen molecule, packaged and secreted into the extracellular space. Subsequently both N- and C-propeptides are cleaved to release a collagen molecule. The collagen molecules are then spontaneously self-assembled into a fibril and stabilized by covalent intra- and intermolecular covalent cross-linking. During fibrillogenesis, molecules are packed in parallel and longitudinally staggered by an axial repeat distance, D period (∼67 nm), creating two repeated regions, that is, the overlap and hole regions, in the fibril.

The origin of collagens—Collagen phylogenetics

The collagen family has 28 unique collagen triple helixes, with 46 different unique polypeptide chains. This suggests that the collagens are a big family of proteins, with conserved functions, that have been refined throughout evolution to serve specific purposes. Which collagen was the first, and are there different, more conserved evolutionary subgroups that harbor specific functions? These are intriguing questions for all collagen researchers. This should be seen in the light of that elastin [92] and fibronectin [93] are essential components of the ECM with only one gene encoding for the polypeptide structures.

Phylogenetics is the study of evolutionary relationships, typically within species, proteins, or genes. By generating a phylogenetic tree, the clustering of different groups can be studied in an effort to study the direction and progression of the evolution and trace back the line of evolution to a common ancestor.

As seen in Fig. 7 of the collagen tree, there are two major branches that split, both of which contain most of the classical basement membrane (green colors) collagens in one branch and all the fibrillar collagens (blue colors) in the other. The longer nodes translate to greater age, which could indicate that the basement membrane collagens developed earlier than the fibrillar ones, while the higher numbers of branch splits for the fibrillar collagens could indicate a more specified function driven by evolution. Furthermore, the location of these families of collagens on different branches is consistent with the different biological functions and locations in which these collagens are found. Interestingly, collagen type VI (α3, α5, and α6 chains) branches out very early, which could indicate that these specific chains of mature collagen type VI have a highly conserved biological function unaltered by evolution and that the basement membrane was the first ECM to evolve [94]. As suggested [94], the basement membrane was first developed as a result of a pericellular ECM, and the interstitial ECM later developed when more structural components held larger structures in place. In addition, the type VI collagen a3 chain spliced out early on. This is interesting because the collagen hormone endotrophin is the propeptide of that chain [14,28,95,96], which suggests unique signaling functions that are very different from the other collagen chains.

This phylogenetic representation of collagen evolution infers that collagens are highly specific molecules serving distinct functions that have evolutionary motifs that confer specific functions related to their collagen family. These distinct motifs may not just be structural components of various tissues but also part of balancing tissue turnover. Of particular interest, collagens have evolved into many different subgroups with 28 unique collagen molecules and 46 different sidechains. In contrast, the essential ECM components elastin [97] and fibronectin [98], each originate from one gene. Consequently, the divergent evolution of the collagens must have important clues. This suggests that we are just beginning to understand collagens and that many more functions will emerge from the different domains that define their relative subtype, such as fibrillar, networking, FACIT, or even membrane collagens [9]. We know that these collagens have very distinct functions, good and bad, in different tissues [9].

Figure 7  The tree was generated by finding reviewed, human, collagen sequences (no isoforms were included) on the UniProt1 website. The sequences were downloaded in the FASTA format and uploaded to the Clustal Omega website2 (version 1.2.4) to generate a multiple sequence alignment file. The generated phylogenetic tree data were downloaded, and the phylogenetic tree was displayed by using FigTree software version 1.4.33. The tree is presented as a midpoint rooted tree and proportionally transformed branches. The classic basement membrane collagens are colored dark green, while the newer basement membrane collagens are colored light green. Likewise, the classic fibrillar collagens are dark blue, and the newer fibrillar collagens light blue (www.uniprot.org; https://www.ebi.ac.uk/Tools/msa/clustalo/; http://tree.bio.ed.ac.uk/software/figtree/).

Table 1

BMP-1, bone morphogenetic protein-1; BRKS, Bruck syndrome; CCS, Cole-Carpenter syndrome; COMP, cartilage oligomeric matrix protein; CRTAP, cartilage-associated protein; CSVD, cerebral small vessel disease; DEB, dystrophic epidermolysis bullosa; FKBP, FK506-binding protein; FOP, fibrodyplasia ossificans progressiva; HSP47, heat shock protein 47; IA, intercranial aneurysm; LH1/2/3, lysyl hydroxylase 1/2/3; MED, multiple epiphyseal dysplasia; OI, osteogenesis imperfecta; P3H1/2, prolyl 3-hydroxylase 1/2; P4H, prolyl 4-hydroxylase; PEDF, pigment epithelium-derived factor; PKD, polycystic kidney disease; PSACH, pseudo achondroplasia; SPARC, secreted protein acidic and cysteine-rich; TANGO1, transport and golgi organization-1 homolog.

Collagen turnover as function of age

Collagen turnover is highly affected by age, which is important for designing and interpreting experimental settings. In fact, type I and II collagen are more than 100-fold higher in 1-month-old animals compared with 6-month-old animals [99]. As illustrated in Fig. 8, modified from [99], collagen turnover is drastically different in animals undergoing remodeling (rebuilding of tissues) in the face of a modeling period during the building of tissues. This is central to interpreting the results from pharmacological models in the tissue turnover space.

This results in three important observations: (1) the matrix composition and quality may be different in older versus younger animals and (2) the relative induction of a response to an insult and pathology in older versus younger animals is higher, which is important when interpreting biomarkers because this provides better contrast as a smaller induction will not be detectable at high expression levels in young animals though will be highly detectable in low turnover situations. This has been reported for many collagen type I and II markers, such as CTX-I and CTX-II [5,100–102]. (3) Much fibrosis research and tissue turnover research have been conducted in younger animals that have a higher capacity for repair and turnover, which may have resulted in both false-negative and false-positive observations [13,99].

Figure 8  Scheme of age-dependent ECM turnover and serum biomarker development at different ages and after pathological remodeling/fibrosis. Modified from Karsdal et al. [99].

Much of the regulation of these collagens is a result of the closure of the growth plate, but other collagens are also affected 1–4-fold. In contrast, interstitial type III collagen is upregulated by 1–2-fold [99], and the basement membrane is upregulated threefold, as evidenced by the biomarkers of type IV collagen formation and degradation. In rats, both type III and IV collagen stabilize 1–2 months after birth [103]. Other collagens, such as type V and VI, are not regulated. Carefully designed experiments and biomarkers are lacking to provide data on the remaining 28 collagens.

Aligning with the studies on rats, in humans, there is a strong age dependency on collagens, which, however, only has been carefully investigated concerning type I and II collagen. Type I and II collagen both level off at the age of 25 and increase after menopause, age 55 +/− 5 years, as a result of the loss of sex hormones, by 100%–150% [100], which corresponds to the loss in bone mineral density [104–106]. This has been well documented in bone biology, where PINP and CTX-I have been used as biomarkers for decades [107–109].

Why laminins?

As collagens, laminins are structural proteins with helical regions, albeit not as stringent as seen in the triple-helical region of collagens. Laminins are interesting biomarkers in a range of chronic diseases associated with ECM tissue turnover [110,111] and are the closest relative to the collagen family. In collagens, the helical domain is made up of a strict building block that consists of three amino acids Gly-X-Y, where X and Y are often proline (Pro) and hydroxyproline (Hyp), respectively [112]. In contrast, the triple-helical structure found in laminins is made of heptads, which have a less strict organization [112]. The heptad structure (abcdefg)n often has hydrophobic residues at positions a and d [113]. An example of the lower level of strictness in the heptad structure is seen when comparing the coiled-coil regions between species. A comparison of the laminin α5 chain from mammals with that of insects (i.e., drosophila melanogaster) revealed that there was only 29% identity, whereas the other domains had up to 60% identity [114]. Thus, the sequence motif does not rely on specific residues but instead depends on the polarity.

Compared with collagens that are present in all compartments of the body, laminins are solely found in the basement membrane [33,34,115]. The basement membrane is an intricate meshwork composed of laminins, collagen IV, nidogens, and sulfated proteoglycans that separate the epithelium, mesothelium, and endothelium from connective tissue [115,116]. Even though the basement membrane consists of the same proteins throughout the body, different isoforms of these combine to form structurally and functionally diverse basement membranes. During the maturation of most basement membranes, the composition of laminins changes. For example, as part of the maturation of the glomerulus, laminin-1 is present in the early stages but is gradually replaced by laminin-10 and -11. In the final stages of maturation, laminin-10 disappears, leaving laminin-11 as the sole laminin in the glomerular basement membrane (GBM) [117].

The crucial role of laminins in the basement membrane is seen during the development of mice embryos, where it is sufficient for the formation of basement membrane-like structures, even in the absence of the other major basement membrane protein, that is, type IV collagen [118]. Furthermore, with the exception of the laminin-α4, -β2, and -γ3 chains, a deficiency of either of the laminin chains leads to early mortality [115]. Even for those that do not cause early mortality, other major complications arise [117,119–122].

Laminins carry out a central role in organizing the intricate meshwork of the basement membranes. This is seen through a wide range of interaction partners, which include dystroglycan, nidogens, syndecans, integrin, heparin, sulfatides, and more [13,117,119–122]. The interaction of laminins with their partners has been shown to mediate signaling. The wide range of interactions, combined with early mortality seen with deficiency of most laminin chains, underlines the essential role of laminins for maintenance of the basement membrane.

Understanding the epitope of a protein

Proteins entail a suite of diverse domains, each with unique functions. Most, if not all, proteins have different functional domains, and additional information can be obtained by deconstructing the domains. Consequently, precision medicine needs to know if the protein, or epitope of the protein, is associated with tissue formation or tissue degradation, protein binding, or signaling. In a simple schematical way, as illustrated in Table 2, one gene may in fact give rise to hundreds of proteins or protein epitopes, each with a unique meaning in the pool of proteins.

Table 2

These proteins are further modified by post-translational modifications (PTMs), which generate protein fragments and modifications with additional functions when compared with the intact native protein. In addition to these PTM, the proteins may contain domains with a unique signaling function that is only realized after protein processing, such as the collagen signaling domains, of which endostatin is the most well-known. Examples of single amino acid PTM are nitrosylation, cross-linking by transglutaminases, acetylation, phosphorylation, hydroxylation, isomerization, glycosylation, and many others.

In addition to the coded protein, a range of important PTMs are found in proteins that would not have been detected by standard protein arrays. Consequently, quantification of total proteins leaves out a range of essential information related to the biological meaning, such as tissue formation or degradation information. In addition to degradation and formation, select enzymes are responsible for targeted single amino acid modifications, resulting in PTM. A selected list of these PTMs includes glycosylation, aging, citrullinations, nitrosylation, cross-linking by transglutaminases, acetylations, phosphorylations, hydroxylations, and others [123], supporting the notion that modified proteins carry additional information compared with their native origins [31], of which many have altered biological functions [123]. In collagens, several crosslinkings by lysyl oxidase are essential for stabilizing the triple helix and thus protein function [124,125]. The most famous example is HbA1c, a glycosylated hemoglobin used as a diagnostic, prognostic, and efficacy intervention biomarker for type II diabetes [126] but which, in its natural forms, indicate anemia or blood cancers. In conclusion, one protein may result in thousands of quantifications of the protein version, all of which have different meanings. These epitopes need to be deconvoluted to provide the best tools for precision medicine; indeed, the quantification of total proteins is just the beginning.

Why do we need to quantify the ECM?

There is a critical need for predictive biomarkers in clinical research. Patients and payers demand greater efficacy and safety windows. Because of scarce research funding, drug developers are forced to select projects they are confident early on in the development process for investment and further investigation in expensive phase III studies. Although the need is clear, the most frequently used methods of quantification within serum or plasma samples are more than 2 decades old. These methods quantify the total proteins and overlook new developments and understandings of proteins as complex players that have multiple functions. Because the body regenerates, there is a need to associate the biomarker with either the degradation or formation in tissues, as exemplified with several novel tissue turnover biomarkers [10,103,127–129]. Quantifying each part of the protein separately may elucidate a wealth of information. Proteins have multiple domains, each with distinct functions [1]. Consequently, if we rely exclusively on total protein assays, we may miss the most essential and important function of a protein that is not regulated but where a fragment contains essential pathological information, such as CTX-II, C3M, or many other well-described protein fragments that have been identified though total protein arrays but classical biochemistry [130]. Collagens contain a variety of domains that can be quantified individually, providing an abundance of information. Some examples are as follows:

Propeptides

The N- and C-terminal ends of fibrillar collagens contain propeptides, which are cleaved and separated from the molecule when the collagens are embedded into the matrix. The mature collagen structure will only be incorporated correctly into the ECM when the propeptides are removed. The maturation and correct processing of collagens are essential for the quality of the structure of the ECM. For decades, the propeptides can be used as a surrogate measure of tissue formation. Biomarkers of type I collagen formation (PINP or PICP) have been used for the assessment of bone formation [101,131]. Only the neoepitope Protein Fingerprint™ technology allows for the assessment of the exact propeptides generated during tissue formation by measuring the cleavage site neoepitope that is produced by proteases during this process. The type I collagen molecule with the epitopes of tissue formation and degradation is illustrated in Fig. 9.

Degradation fragments

To sustain healthy tissues, the ECM is continuously being remodeled; as such, the associated ECM proteins are degraded. This degradation results in the release of small protein fragments from larger complex proteins; these circulating fragments may be used as biomarkers. These smaller protein fragments contain information related to specific tissue degradation. For example, bone is continuously remodeled by bone-degrading osteoclasts. The main destructive protease responsible is cathepsin K. The cleavage of type I collagen by cathepsin K yields the fragment referred to as CTX-I, which has been used as a biomarker of bone resorption for diseases in the osteology field [2,101,132].

Tissue balance

By assessing the formation and degradation of tissue separately, a greater understanding of tissue homeostasis can be obtained. Traditional protein measurement technologies do not allow for the separate assessment of formation and degradation; rather, these methods quantify the total protein pool. In many diseases, it is the balance between the degradation and formation of tissues that is affected, leading to either more nonfunctional tissue such as in skin, liver, lung, and kidney fibrosis, or increased degradation, leading to tissue loss in rheumatologic disorders. This balance may be severely altered, leading to the same amount of that protein in the blood but with an altered composition. In chronic obstructive pulmonary disease (COPD), 50% less formation and 50% more degradation is observed during exacerbations [133,134]. This imbalance is not detected by traditional assays but only by using specific post-translational modifications [135,136]. This balance is illustrated in Fig. 10, in which a change in the total amount of ECM in a tissue is described by the ratio between formation and degradation. This ratio changed the bone field and resulted in two types of treatments that provided more and better bone mass, either treatment blocking bone resorption (anti-resorptive) or stimulating bone formation (anabolics).

Figure 9  Biomarkers of collagen type I. Schematic representation of type the fibrillar type I collagen, with the N- and C-terminal propeptides that may be used as biomarkers for tissue formation. The triple helical region with type I collagen degradation biomarkers that may be used as biomarkers for tissue degradation.

Signaling peptides

The ECM proteins are emerging as more than just passive structural proteins [137–139]. During tissue remodeling, an array of potent signaling molecules are generated as propeptides during tissue formation and the degradation of the fragments of collagens consequent to tissue degradation. We are only now beginning to understand the meaning behind these signals. Collagens and other proteins contain sequences with potent signaling functions; these sequences become active when they are exposed and released by proteolytic cleavage. One central motif of these molecules is the NC1 domain of type IV [139], VIII [138], XV [140], and XVIII [141] collagen. It is the processing of the NC1 domain that results in these highly potent and well-documented antiangiogenetic fragments that may have therapeutic implications. In addition, the propeptide of type VI collagen, PRO-C6/endotrophin, is receiving a lot of interest, both as a biomarker that is prognostic for IPF [142], SSC, NASH, CVD [143], and kidney outcome but also for the signaling potential as a drug target [96,144]. Assays for the bioactive fragments of these proteins have been developed, as shown in Table 3.

Figure 10  Tissue balance. Schematic figure of the tissue balance, in which the turnover of the total protein may not be altered in a pathology. In contrast, when different subpools of such protein are quantified biomarkers of tissue formation and degradation, this balance may be altered. This schematic highlights the importance of knowing the exact epitope and meaning of that epitope when quantifying biomarkers in modern clinical chemistry.

Table 3

Conclusion

The lessons learned are that a protein is not just a protein and that serological assessments of protein fragments, not a pool of the entire protein, may contain essential information for data interpretation [1]. We can only advance science and become more patient centric is we accurately quantify separately the ECM signaling peptides, tissue formation, and tissue degradation fragments to understand biological processes, so that we may change them pharmacologically. By using more advanced protein assessment technologies, more accurate diagnostic and prognostic information can be obtained.

In this book, we hope to reveal