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

Biochemistry of Collagens, Laminins and Elastin

Structure, Function and Biomarkers

Second Edition

Editor

Morten A. Karsdal

Nordic Bioscience, Herlev, Denmark

University of Southern Denmark, Odense, Denmark

Co-editors

Diana J. Leeming

Kim Henriksen

Anne-Christine Bay-Jensen

Signe Holm Nielsen

Cecilie L. Bager

Table of Contents

Cover image

Title page

Copyright

List of contributors

Foreword

Preface

Acknowledgments

List of abbreviations

Introduction

Chapter 1. Type I collagen

Summary

Chapter 2. Type II collagen

Summary

Biomarkers of type II collagen

Chapter 3. Type III collagen

Summary

Biomarkers of type III collagen

Chapter 4. Type IV collagen

Summary

Biomarkers of type IV collagen

Chapter 5. Type V collagen

Summary

Biomarkers of type V collagen

Chapter 6. Type VI collagen

Summary

Biomarkers of type VI collagen

Chapter 7. Type VII collagen

Summary

Biomarkers of type VII collagen

Chapter 8. Type VIII collagen

Summary

Biomarkers of type VIII collagen

Chapter 9. Type IX collagen

Summary

Biomarkers of type IX collagen

Chapter 10. Type X collagen

Summary

Biomarkers of type X collagen

Chapter 11. Type XI collagen

Summary

Chapter 12. Type XII collagen

Summary

Chapter 13. Type XIII collagen

Summary

Biomarkers of type XIII collagen

Chapter 14. Type XIV collagen

Summary

Chapter 15. Type XV collagen

Summary

Chapter 16. Type XVI collagen

Summary

Biomarkers of type XVI collagen

Chapter 17. Type XVII collagen

Summary

Chapter 18. Type XVIII collagen

Summary

Tissue expression

The 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

Summary

Biomarkers of type XIX collagen

Chapter 20. Type XX collagen

Summary

Chapter 21. Type XXI collagen

Summary

Biomarkers of type XXI collagen

Chapter 22. Type XXII collagen

Summary

Biomarkers of type XXII collagen

Chapter 23. Type XXIII collagen

Summary

Biomarkers of type XXIII collagen

Chapter 24. Type XXIV collagen

Summary

Chapter 25. Type XXV collagen

Summary

Chapter 26. Type XXVI collagen

Summary

Chapter 27. Type XXVII collagen

Summary

Chapter 28. Type XXVIII collagen

Summary

Chapter 29. Laminins

Summary

Introduction

Structure

Nomenclature

Interaction partners

Signaling

Laminin trimers

Laminin mutations and their disease in man

Chapter 30. Elastin

Summary

Chapter 31. The collagen chaperones

Summary

Chaperone introduction

SPARC

Periostin

COMP

HSP47

Chapter 32. Collagen diseases

Summary

Osteogenesis imperfecta

Ehlers–Danlos syndrome

Alport’s syndrome

Epidermolysis bullosa

Chapter 33. The signals of the extracellular matrix

Summary

Introduction

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

Type VI collagen—endotrophin

Type VIII collagen—vastatin

Type XV collagen—restins

Type XVIII collagen—endostatin

Other extracellular matrix fragments

Chapter 34. The roles of collagens in cancer

Summary

Desmoplasia

Collagen binding to receptors regulate cancer cell fate and metastasis

Altered degradation of collagen in the tumor

Collagen fragments as noninvasive biomarkers of desmoplasia

Chapter 35. Use of extracellular matrix biomarkers in clinical research

Summary

Biomarker terminology

Extracellular matrix biomarkers as a translational tool in drug development

Established use of ECM biomarkers in drug development and epidemiology

Potential for future use

Chapter 36. Common confounders when evaluating noninvasive protein biomarkers

Summary

Technical and analytical validation of novel noninvasive protein biomarkers

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 37. Implementation of collagen biomarkers in the clinical setting

Summary

How to implement biomarkers in clinical development

Definitions of biomarkers in healthcare and drug development

Biomarkers used to increase knowledge on whom to treat and derisk phase III programs

Biomarkers used for enrichment of trial populations—the qualification route

Discussion

Index

Copyright

Academic Press is an imprint of Elsevier

125 London Wall, London EC2Y 5AS, United Kingdom

525 B Street, Suite 1650, San Diego, CA 92101, United States

50 Hampshire Street, 5th Floor, Cambridge, MA 02139, United States

The Boulevard, Langford Lane, Kidlington, Oxford OX5 1GB, United Kingdom

Copyright © 2019 Elsevier Inc. All rights reserved.

No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, recording, or any information storage and retrieval system, without permission in writing from the publisher. Details on how to seek permission, further information about the Publisher’s permissions policies and our arrangements with organizations such as the Copyright Clearance Center and the Copyright Licensing Agency, can be found at our website: www.elsevier.com/permissions.

This book and the individual contributions contained in it are protected under copyright by the Publisher (other than as may be noted herein).

Notices

Knowledge and best practice in this field are constantly changing. As new research and experience broaden our understanding, changes in research methods, professional practices, or medical treatment may become necessary.

Practitioners and researchers must always rely on their own experience and knowledge in evaluating and using any information, methods, compounds, or experiments described herein. In using such information or methods they should be mindful of their own safety and the safety of others, including parties for whom they have a professional responsibility.

To the fullest extent of the law, neither the Publisher nor the authors, contributors, or editors, assume any liability for any injury and/or damage to persons or property as a matter of products liability, negligence or otherwise, or from any use or operation of any methods, products, instructions, or ideas contained in the material herein.

Library of Congress Cataloging-in-Publication Data

A catalog record for this book is available from the Library of Congress

British Library Cataloguing-in-Publication Data

A catalogue record for this book is available from the British Library

ISBN: 978-0-12-817068-7

For information on all Academic Press publications visit our website at https://www.elsevier.com/books-and-journals

Publisher: Andre G. Wolff

Acquisition Editor: Linda Versteeg-buschman

Editorial Project Manager: Sandra Harron

Production Project Manager: Poulouse Joseph

Cover Designer: Christian J. Bilbow

Typeset by TNQ Technologies

List of contributors

J.R. Andersen,     Nordic Bioscience, Herlev, Denmark

A. Arvanitidis,     Nordic Bioscience, Herlev, Denmark

C.L. Bager,     Nordic Bioscience, Herlev, Denmark

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

A.R. Bihlet,     Nordic Bioscience, Herlev, Denmark

A. Engstroem,     Nordic Bioscience, Herlev, Denmark

E. Erhardtsen,     Nordic Bioscience, Herlev, Denmark

F. Genovese,     Nordic Bioscience, Herlev, Denmark

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

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

K. Henriksen,     Nordic Bioscience, Herlev, Denmark

Y. He,     Nordic Bioscience, Herlev, Denmark

C. Jensen,     Nordic Bioscience, Herlev, Denmark

H. Jessen,     Nordic Bioscience, Herlev, Denmark

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

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

A. Kerrn-Jespersen,     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

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

M. Lindholm,     Nordic Bioscience, Herlev, Denmark

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

T. Manon-Jensen,     Nordic Bioscience, Herlev, Denmark

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

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

S. Holm Nielsen,     Nordic Bioscience, Herlev, Denmark

N.I. Nissen,     Nordic Bioscience, Herlev, Denmark

M. Pehrsson,     Nordic Bioscience, Herlev, Denmark

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

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

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

J.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

C.S. Thudium,     Nordic Bioscience, Herlev, Denmark

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

N. Willumsen,     Nordic Bioscience, Herlev, Denmark

Foreword

It is with great pleasure that I write this foreword for the book Biochemistry of Collagens, Laminins and Elastin by Morten Karsdal and colleagues. I was raised as a collagen researcher in those times when collagen and matrix research started to virtually explode, with the structural and functional elucidation of the fibril-forming collagens types I, II, III, and V, basement membrane collagen type IV, and the microfilamentous collagen type VI, accompanied by the discovery and characterization of laminin A, nidogen/entactin, and some proteoglycans. In those times, I was lucky to do my diploma and PhD thesis in the Institute of Connective Tissue Research at the Max-Planck-Institute for Biochemistry in Munich, with my mentors Rupert Timpl and Klaus Kühn, and within a large community of national and international top-notch matrix researchers. Notably, these discoveries were purely based on (protein) biochemistry, a very labor-intensive enterprise. Just then DNA sequencing became available, still at a pace unbelievably slow compared to today’s technologies. Although gene sequencing dramatically enhanced our knowledge about the multitude of matrix proteins, enzymes, receptors, and regulators, this came at a cost, namely a dwindling community of protein biochemists working on matrix proteins, especially collagens. The dramatic advance in discovering novel matrix genes had fallen behind their functional characterization for many years. However, the group of matrix and collagen experts that now combine the genetic information with protein chemistry and functional studies is growing again, as exemplified by the unique assembly of experts and expertise in the present book. Thus each of the 28 collagen types that are all trimeric molecules composed of the same or variant chains of the 46 different collagen chains are described and visually presented in all detail, as far as current knowledge allows. Importantly, a major focus of this book is also on functional and translational aspects of the different collagens and select glycoproteins and proteoglycans that are only partly predictable by genetic sequencing. These aspects involve 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 at the verge of being exploited for novel therapies to address organ fibrosis, inflammation, and cancer. Moreover, the utility of serum markers that derive from specific matrix components and their defined naturally produced proteolytic fragments, are outlined in detail. This is a rapidly evolving field that will permit a noninvasive assessment of the dynamics of matrix synthesis or degradation in an organ- and disease-specific way. The availability of such surrogate markers has already begun to spur specific antifibrotic drug development in clinical studies for liver, lung, and kidney fibrosis that otherwise have to be based on sequential biopsies or difficult-to-interpret functional readouts.

There is no single review nor assembly of reviews that covers all these areas of collagen and matrix research in a comparably comprehensive way and across narrow professional boundaries, as this book does. I consider it the best available review that will be an invaluable resource for every researcher and clinician with an interest in matrix biology, biochemistry, and translational perspectives.

Detlef Schuppan, MD, PhD,     Professor of Medicine, Mainz University Medical Center and Harvard Medical School

Preface

This book on extracellular matrix proteins is made consequent to the love of matrix biology and structural proteins. Controlling the life, death, and fate of cells, extracellular matrix proteins emerge as much more than passive bystanders in this fascinating cycle.

The physiological and pathophysiological role and function of many other collagens remain to be discovered and presented. We hope this book will inspire new researchers to take on the collagen challenge and present novel research and biology crucial for the understanding of the extracellular matrix in pathological and physiological conditions.

Many researchers and important works have been cited. However, not all who deserve to be cited for their work have been cited. This is a request for everyone working on collagens, laminins, and elastin to please send us your reference and a summary of the work to be included in future editions of this book. We aspire to have this book as complete as possible for collagen research biomarkers and biology. Please help us with that aspiration.

Sincerely

Morten A. Karsdal, MSc, PhD, mMBA,     Professor, SDU

Acknowledgments

I wish to thank Claus Christiansen for discovering, developing, and FDA-validating the first biomarker of extracellular matrix (ECM) remodeling, CTX-I; a fantastic fragment made by cathepsin K of type I collagen, now recognized as the standard bone resorption marker. This discovery has inspired many researchers, including myself, to uncover, develop, and validate biomarkers of the ECM. 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 present and former PhD students as well as senior researchers that have helped me understand and quantify the matrix. Without your dedication and hard work in generating assays and data, this book would have been impossible. Further special thanks go to all the excellent technical help 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 the help with the book.

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

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

Sincerely

Morten A. Karsdal

List of abbreviations

97-LAD   97-kDa linear IgA dermatosis antigen

aa   Amino acid

ADAM   A disintegrin and metalloproteinase

ANCA   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   180-kDa bullous pemphigoid antigen

BP230   230-kDa 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

ISEMF   Intestinal subepithelial myofibroblasts

JEB   Junctional epidermolysis bullosa

KO   Knock-out

LAD-1   120-kDa linear IgA dermatosis antigen

LG   Laminin globular

MI   Myocardial infarction

MIM   Mendelian inheritance in man

MMP   Matrix metalloproteinases

mRNA   Messenger ribonucleic acid

MTJ   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

α1   α1 chain

α2   α2 chain

Introduction

M.A. Karsdal

The backbone of tissues is composed of structural proteins such as collagens, elastin, and laminins. During tissue turnover, these 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. 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. Even more importantly the proteins are also orientated differently. Thus, fibrosis is both a protein accumulation and disorientation/mallocalization pathology. In fact, a good protein (collagen) may be a bad collagen in different places [1,2]—and consequently, the exact function of the individual collagens becomes very important.

The core protein of fibrosis is collagen and other structural proteins such as laminins and elastin. Collagens are not just collagens, and each collagen has unique expression patterns. Some collagens not only have simple structural functions but are also key for signaling. 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 essentials of collagen biology, expression, and function.

Tissue remodeling is essential for sustaining a healthy tissue. Extracellular (ECM) remodeling, in which old or damaged molecules are degraded and replaced by new ones, is a key process in tissue homeostasis. A well-thought-out example of tissue homeostasis is the bone, once believed to be a static tissue, which is now known to be regenerated completely every 10–25  years, depending on location and bone type, i.e., cortical vs. trabecular, respectively [3]. Remodeling of bone is a dynamic process requiring a closely orchestrated interaction between the cells of bone and the ECM of the bone. When this tight equilibrium is out of control, such as in cancer and osteomalacia, the result is a dysregulated remodeling leading to fibrotic or woven bone, with a disorganized structure that lacks the mechanical strength of normal bone [3,4].

Why are collagens and structural proteins important?

Fibrosis can affect almost any organ or tissue, albeit in different ways [5,6]. Fig. 1 illustrates the major fibroproliferative diseases with a significant impact on human health. 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 [7,8]. Fibrosis can result either from injury to tissue, in which case it manifests as scarring, or from abnormal connective tissue turnover [2].

Forty-five percent of all deaths in the developed world are associated with chronic fibroproliferative diseases [5], 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 excessive and abnormal accumulation of ECM components in the affected tissues and ultimately tissue failure [10]. This ECM has an altered structure and signals abnormally to the cells that are embedded in it [1,11]. The ECM composition changes dramatically during the development of fibrosis as the proteins interact with each other and the cells that attach to them [2].

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. 

(Inspired by Refs. [1,9].)

Fibrotic tissue was for a long time considered an inactive scaffold, preventing regeneration of the affected organ. However, this perception cannot be upheld since fibrosis is neither static nor irreversible, but instead the result of a continuous remodeling process and thereby susceptible to intervention [5,6,12,13]. The future challenge in fibrosis will be to halt fibrogenesis and reverse advanced fibrosis without affecting tissue homeostasis or interfering with normal wound healing [14–17]. 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.

Traditionally, only growth factors, cytokines, hormones, and other small molecules have been considered as relevant mediators of inter-, para- and intracellular communication and signaling. However, the ECM fulfills direct and indirect paracrine or even endocrine roles [18]. 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 [2]. 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 [19]. This and other collagen fragments will be discussed. 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 [20–22].

This book aims to: (1) summarize available data of key structural proteins of the ECM (i.e., collagens, laminins, and elastin); (2) review how these molecules affect pathologies, in part, exemplified by monogenetic disorders; (3) describe selected posttranslational modifications (PTMs) of ECM proteins that result in altered signaling properties of the original ECM component and that these collagens harbor cryptic signaling functions that may be viewed as endocrine functions; (4) focus on the common confounders for serological measurements; (5) discuss the important collagen-binding and -modulating proteins—the collagen chaperones; (6) discuss the use of collagen biomarkers in clinical research; (7) discuss the collagen diseases; and (8) discuss whether we may use collagens and regulatorily approved biomarkers.

Introduction to the matrix—interstitial and basement membranes

There are two main ECM areas in the body: the basement membrane and the interstitial matrix. The basement membrane is underlying endothelium and epithelium, holding these cells in a permeable loose matrix ensuring that the cells are polarized and functionable. The main constituents of the basement membrane are type IV collagen, nidogen, and laminins [20,23–26]. The interstitial matrix is responsible for the structure and rigidity of the tissues, mainly produced by fibroblasts [2,27]. The primary proteins in the interstitial matrix are collagens produced mostly by fibroblasts. The main collagens of the interstitial matrix are types I, III, V, and VI [28].

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). Tumstatin, which is a specific fragment of collagen type IV, is a perfect example of the cell-regulatory potential of the ECM molecules. Tumstatin has been shown to be very antiangiogenic, possibly directing recovery of the epithelium by allowing horizontal growth over the basement membrane rather than uncontrolled vertical growth into the basement and interstitial membranes. In the transition area between the basement membrane and intestinal interstitial matrix, other collagens such as type XVIII are present, with other functions. A protease-derived fragment of collagen type XVIII, endostatin [29], is the most potent natural antiangiogenic molecule which has been shown to block fibrosis in fibrotic models of the liver and lung. Although it needs to be investigated [2] other collagens, such as type XV, may play similar or more tissue-specific roles by releasing active protein fragments (called neoepitopes) such as restin. During repair of the matrix, following epithelial damage, the underlying membranes are destroyed by proteases, giving rise to new signaling molecules which may be both antifibrogenic and antiangiogenic and could potentially have other functions that are yet to be discovered.

The deeper intestinal matrix consists mainly of types I and III collagen, which are produced by fibroblasts and responsible for the rigid structure of the ECM, allowing tissue function [11,30–32]. Recently, however, the minor collagens produced by fibroblasts have been shown to have signaling potential, such as the newly discovered fragment of the propeptide of type VI collagen, endotrophin [18,33]. Consequently, the deep dense intestinal matrix is also more than a passive bystander, which needs to be taken into account in healthy and pathological situations.

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 in the body. 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 [34]. Fig. 3 schematically displays the primary structure of the molecules. Collagens are trimeric molecules composed of three polypeptide α-chains which contain the repeated sequence (G–X–Y)n, X being frequently proline and Y hydroxyproline. These repeats allow 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), which is secreted and deposited into the ECM. Most collagens can form supramolecular aggregates. Besides triple-helical domains, collagens contain nontriple-helical (NC) domains, used as building blocks by other ECM proteins. The molecular structure and supramolecular assembly of collagens allow their division into major subfamilies, depending on the supramolecular structure as depicted in Fig. 4.

Figure 2  Schematic representation of the generation from the basement membrane and interstitial membrane of biomarkers of endothelial or epithelial cell damage. (A) Overview of the endothelial cell layer which has 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 result in overproduction of components of the interstitial and basement membranes in an unorganized manner, i.e., fibrosis (D2).

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

Figure 4  The supermolecular structure of collagens.

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

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

3. Network-forming collagens (IV) 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. Hexagonal network forming collagens (VII and X);

5. Beaded filaments forming collagens (VI);

6. Anchoring fibrils (collagen VII).

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. 5. This complex process is mediated by 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 crosslinks leading to the final and mechanically competent collagen fibrils. Further details on the molecular aspects of this process are outside the scope of this chapter, but we refer the reader to Refs. [35–38]. 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 posttranslational modifications. In the synthesis process, the heat shock protein 47 (HSP47) binds to procollagen in the endoplasmic reticulum. HSP47 is a specific molecular chaperone of procollagen [39]. The stabilization of the procollagen triple helix at body temperature requires the binding of more than 20 HSP47 molecules per triple helix [26]. 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 [40]. In direct alignment, cleavage of SPARC was shown to increase collagen affinity and protect against MMP degradation of fibrillar collagens [41]. Both propeptides of the N and C terminal of 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 Bone Morphogenetic Protein-1 (BMP-1) [42–44]. 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 step releasing the propeptides from the mature intact collagen triple helix and allowing it 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 [36]. Lastly, the telopeptides contain the sites where crosslinking occurs. This process is initiated by the oxidative deamination of lysyl and hydroxylysyl residues catalyzed by the enzymes of the lysyl oxidase family.

Figure 5  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-αchains in the ER specific peptidyl lysine residues are hydroxylated to form hydroxylysine (–OH–NH 2 ) and, subsequently, specific glycosylated hydroxylysine residues, this latter step being called O-linked glycosylation. For the latter, either single galactose (a green hexagon ) or glucose-galactose ( red and green hexagons ) are attached. After these and other modifications (for example, hydroxylation of proline, or asparagine-linked glycosylation), two pro-α1 chains and one pro-α2 chain 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 crosslinking. 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 whole regions, in the fibril.

The origin of the collagens—collagen phylogenetics

Which collagens were the first, and are there different more conserved evolutionary subgroups?

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 to study the direction and progression of the evolution and trace back the line of evolution to a common ancestor.

As seen in Fig. 6 of the collagen tree, there are several branch splits, one containing most of the classical basement membrane (green colors) collagens and another containing 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 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 the mature collagen type VI have a highly conserved biological function unaltered by evolution.

Figure 6  The tree was generated by finding reviewed, human, collagen sequences (no isoforms were included) on the UniProt [74] Website. The sequences were downloaded in the Fasta format and uploaded to the Clustal Omega website [75] (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 1.4.3 [76] . 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.

This phylogenetic representation of collagen evolution infers that collagens are highly specific molecules serving distinct functions, and not just structural components of various tissues.

Collagen turnover as function of age

Collagen turnover is highly affected by age, which is important for designing and interpretation of experimental settings. In fact, types I and II collagen are more than 100-fold higher in 1-month-young animals as compared to 6-month-old animals [45]. As illustrated in Fig. 7, modified from Ref. [45], collagen turnover is drastically different in animals undergoing remodeling (rebuilding of tissues) in the face of the modeling period, during the building of tissues.

This results in three important observations: (1) the matrix composition and quality may be different in older versus younger animals; (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 as this provides better contrast as a smaller induction will not be detectable at high expression levels in young animals, albeit highly detectable in low-turnover situations. This has been reported for many collagen types I and II markers such as CTX-I and CTX-II [46–50]. (3) Much fibrosis research and tissue turnover research have been conducted in younger animals which have a higher capacity for repair and turnover, which may have resulted in both false-negative but also false-positive observations [1,45].

Figure 7  Scheme of age-dependent ECM turnover and serum biomarker development at different ages and after pathological remodeling/fibrosis. 

Reproduced with permission from Hansen JF, Juul Nielsen M, Nyström K, Leeming DJ, Lagging M, Norkrans G, et al. PRO-C3: a new and more precise collagen marker for liver fibrosis in patients with chronic hepatitis C. Scand J Gastroenterol January 2018;53(1):83–7.

Much of the regulation of these collagens is consequent to the closure of the growth plate, but also other collagens are affected one- to fourfold. In contrast, the interstitial type III collagen is upregulated by one- to twofold [45], and basement membrane is upregulated threefold [45], as is evident by biomarkers of type IV collagen formation and degradation. Both types III and IV collagen stabilize 1–2  months after birth in rats. Other collagens such as types V and VI are not regulated. Carefully designed experiments and biomarkers are lacking to provide data on the remaining 28 collagens.

In direct alignment, in man, there is a strong age dependency on collagens, which however only has been carefully investigated concerning types I and II collagen. Types I and II collagen level off at the age of 25, and increase after menopause, age 55  ±  5  years, consequent to the loss of sex hormones by 100%–150% [51], which corresponds to the loss in bone mineral density [49,52–54]. This is well documented in bone biology, where PINP and CTX-I have been used as biomarkers for decades [55–57].

Why laminins?

Laminins as collagens are structural proteins with helical regions, albeit not as stringent as seen in the triple helical region of collagens. They are, however, the closest relative to the collagen family. In collagens the helical domain is made up of a strict building block which consists of three amino acids Gly-X-Y, where X and Y are often proline (Pro) and hydroxyproline (Hyp), respectively [58]. In contrast, the triple helical structure found in laminins is made of heptads which have a less strict organization [58]. The heptad structure represented (abcdefg)n often has hydrophobic residues at positions a and d [59]. An example of the lower level of strictness in the heptad structure is seen when comparing the coiled-coil regions between species. Comparison of the laminin α5 chain from mammals with that of insects (that is, Drosophila melanogaster) revealed that there was only 29% identity, whereas the other domains had up to 60% identity [60]. It thus seems that the sequence motif does not rely on specific residues, but rather depends on the polarity.

Compared to collagens that are present in all compartments of the body, laminins are solely found in the basement membrane [23,24,61]. The basement membrane is an intricate meshwork composed of laminins, collagen IV, nidogens, and sulfated proteoglycans, which separate the epithelium, mesothelium, and endothelium from connective tissue [61,62]. 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) [63].

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, type IV collagen [64]. Furthermore, except for the laminin α4, β2 and γ3 chains, deficiency of either of the laminin chains leads to early lethality [61]. Even for those that do not cause early lethality other major complications arise [63,65–68].

Laminins carry out a central role in organizing the intricate meshwork of the basement membranes. This is seen through the wide range of interaction partners which include dystroglycan, nidogens, syndecans, integrin, heparin, sulfatides, and more [1,63,65–68]. The wide range of interactions combined with early lethality seen with deficiency of most laminin chains underlines the necessity of laminin presence in the basement membrane. In general, the essential role of laminins for maintenance of the basement membranes is exemplified by the lethality of most null mutations.

Why biomarkers?

There is a critical need for predictive biomarkers in clinical research. Patients and payers demand greater efficacy and safety windows. Due to scarce research funding, drug developers are forced to select projects they are confident in early on in their development for investment and further investigation in expensive phase III studies. While the need is clear, the most frequently used methods of quantification within serum or plasma samples are more than two decades old. These methods quantify total proteins and overlook new developments and understanding of proteins as complex players that have multiple functions. Quantifying each part of the protein separately may elucidate a wealth of information. Proteins have multiple domains, each with distinct functions. The collagens contain a variety of domains that can be quantified individually, providing an abundance of information. Some examples are:

1. The 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 the correct processing of the collagens is essential for the quality of the structure of the ECM. 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 assessment of bone formation [49,69] for decades (Fig. 8). Only the neoepitope Protein Fingerprint technology allows for the