Optimized Methods for the Surface Immobilization of Collagens and Collagen Binding Assays

Summary

This work presents an optimized protocol to reproducibly immobilize and quantify type I and III collagen onto microplates, followed by an improved in vitro binding assay protocol to study collagen-compound interactions using a time-resolved fluorescence method. The subsequent step-by-step data analysis and data interpretation are provided.

Abstract

Fibrosis occurs in various tissues as a reparative response to injury or damage. If excessive, however, fibrosis can lead to tissue scarring and organ failure, which is associated with high morbidity and mortality. Collagen is a key driver of fibrosis, with type I and type III collagen being the primary types involved in many fibrotic diseases. Unlike conventional protocols used to immobilize other proteins (e.g., elastin, albumin, fibronectin, etc.), comprehensive protocols to reproducibly immobilize different types of collagens in order to produce stable coatings are not readily available. Immobilizing collagen is surprisingly challenging because multiple experimental conditions may affect the efficiency of immobilization, including the type of collagen, the pH, the temperature, and the type of microplate used. Here, a detailed protocol to reproducibly immobilize and quantify type I and III collagens resulting in stable and reproducible gels/films is provided. Furthermore, this work demonstrates how to perform, analyze, and interpret in vitro time-resolved fluorescence binding studies to investigate the interactions between collagens and candidate collagen-binding compounds (e.g., a peptide conjugated to a metal chelate carrying, for example, europium [Eu(III)]). Such an approach can be universally applied to various biomedical applications, including the field of molecular imaging to develop targeted imaging probes, drug development, cell toxicity studies, cell proliferation studies, and immunoassays.

Introduction

The accumulation of fibrous connective tissue as part of the natural wound-healing process following tissue injury is known as fibrosis. However, if the deposition of fibrous tissue fails to terminate and continues beyond what is needed for tissue repair, then fibrosis becomes excessive^1,2. Excessive fibrosis impairs organ physiology and function and could lead to organ damage and potentially organ failure^3,4,5. Two main drivers of fibrosis are the extracellular matrix (ECM) proteins collagen type I and type III⁶. Collagen is a structural protein found in various organs that makes up approximately one-third of the total protein content of the human body¹. There are 28 different types of collagens identified by human genome sequencing, and the most abundant are the fibrillar collagens⁷. The primary fibrillary collagen is type I collagen, which provides the ECM with tensile strength and resistance to deformation⁸. Type III collagen is a structural component that provides elasticity and colocalizes with type I collagen. It is expressed during embryogenesis and is naturally found in small amounts in adult skin, muscle, and blood vessels⁹.

In vivo collagen synthesis begins with an intracellular process in which mRNA is transcribed in the nucleus and then moves to the cytoplasm, where it is translated. After translation, the chain formed undergoes post-translational modification in the endoplasmic reticulum, where pro-collagen (the precursor of collagen) is formed. Pro-collagen then travels to the Golgi apparatus for final modification before being excreted to the extracellular space¹⁰. Through proteolytic cleavage, pro-collagen is transformed into tropocollagen. This is then cross-linked either via an enzymatic-mediated cross-linking pathway catalyzed by the enzyme lysyl oxidase (LOX) or via a non-enzymatic-mediated cross-linking pathway involving the Maillard reaction¹¹. In vitro protocols to immobilize collagen mainly rely on the ability of collagen to self-assemble. Collagen is extracted from tissues based on its solubility, which largely depends on the extent of cross-linking of individual collagen fibrils⁷. Fibrillar collagen is dissolved in acetic acid, and fibrils can reform when the pH and temperature are adjusted¹². In vitro, the fibrillogenesis of collagen can be viewed as a two-stage process⁷. The first stage is the nucleation phase, where collagen fibers form dimers and trimer fibrils before they are rearranged to form a triple helical structure. The second phase is the growth phase, where the fibrils start to grow laterally and result in the characteristic D-band formation, which is generally observed by changes in turbidity⁷. Atomic force microscopy (AFM) studies have also revealed that type I and type III collagen have different characteristics (Table 1)¹³.

To study the binding interactions between collagen and other compounds, collagen needs to be reproducibly immobilized into the wells of microplates. There are various protocols for immobilizing soluble collagen^14,15,16. Commercially available microplates that are pre-coated with collagen are typically used for cell culture. However, pre-coated microplates have a very thin layer of an unknown amount of collagen coated onto the wells, which makes them unsuitable for in vitro binding assays. There are several challenges when immobilizing collagen onto the plate wells. One of the key challenges is choosing a suitable type of microplate, because different types of collagens (e.g., type I and III) have different chemical properties and, therefore, immobilize more stably and effectively depending on the material of the microplate. Another challenge is the experimental conditions of the immobilization protocol, as the process of fibrillogenesis depends on multiple factors, including temperature, pH, the stock concentration of collagen, and the ionic concentration of the buffer⁷.

For studying the interactions between the collagen (the target) and other compounds (i.e., a targeting peptide), it is also necessary to develop a robust screening assay to investigate the specificity and selectivity of the compound toward the target by measuring the dissociation constant, K_d. The position of the equilibrium of formation of a bimolecular complex between a protein (collagen) and a ligand is expressed in terms of the association constant K_a, whose magnitude is proportional to the binding affinity. However, most commonly, biochemists express affinity relationships in terms of the equilibrium dissociation constant, K_d, of the bimolecular complex, which is defined as K_d = 1/K_a (K_d and is the inverse of K_a).The lower the K_d value, the stronger the binding strength between the protein and the ligand. The advantage of using K_d to compare the binding affinity of different ligands for the same protein (and the other way around) is linked to the fact that the units of K_d for a bimolecular complex are mol/L (i.e., concentration unit). Under most experimental conditions, the K_d value corresponds to the ligand concentration that leads to 50% saturation of the available binding sites on the target at the equilibrium^17,18. The dissociation constant is typically extracted by analyzing the receptor fractional occupancy (FO), which is defined as the ratio between the occupied binding sites and total available binding sites, as a function of ligand concentration. This can be done provided that an analytical assay able to distinguish and measure the amount of bound ligand is available.

In vitro ligand binding assays can be performed using various bioanalytical methods, including optical photometry, radioligand methods, inductively coupled plasma mass spectrometry (ICP-MS), and surface plasmon resonance (SPR). Amongst the photometric methods, those based on fluorescence emission typically require the labeling of ligands or proteins with fluorophores to increase the sensitivity and to improve the detection limit of the assay. Chelates of certain lanthanide(III) ions, such as Eu(III), are very attractive as fluorophores as they have large Stokes' shifts, narrow emission bands (providing a good signal-to-noise ratio), limited photobleaching, and long emission lifetimes. Importantly, the latter property enables the use of time-resolved fluorescence (TRF) from Eu(III) fluorophores to abolish background autofluorescence¹⁹. In the dissociation-enhanced lanthanide fluorescent immunoassay (DELFIA) version of the Eu(III)-based TRF assay, ligands labeled with a non-luminescent Eu(III)-chelate are incubated with the receptor immobilized onto microplates. The labeled ligand/receptor complex is separated from the unbound ligand, and Eu(III) fluorescence is activated by dissociation of the Eu(III) complex at an acidic pH, followed by re-complexation with a fluorescence-enhancing chelator to form a micelle-embedded, highly fluorescent Eu(III) complex²⁰.

The decomplexation step can be reasonably achieved with chelators, such as diethylenetriamine pentaacetate (DTPA), that show fast decomplexation kinetics. However, Eu(III) complexes with certain macrocyclic chelators, such as DOTA (1,4,7,10-tetraazacyclododecane1,4,7,10-tetraacetic acid) and its monoamide derivatives (DO3AAm), show high thermodynamic stability and very high kinetic inertness. In this case, the decomplexation steps must be accurately optimized to achieve sufficient and reproducible activation of Eu(III)-based TRF²¹. It is worth noting that lanthanide (Ln(III))-DOTA and Ln(III)-DO3AAm complexes are those most commonly employed as contrast agents for in vivo molecular imaging by magnetic resonance imaging (MRI) techniques²². Thus, the Ln(III)-based TRF assay is the tool of choice to study in vitro the binding affinity of MRI molecular probes with their intended biological targets. Currently, comprehensive and reproducible protocols for immobilizing type I and type III collagen and a reproducible pipeline for performing in vitro binding Eu(III) TRF experiments are lacking. To overcome these limitations, reproducible methods to self-assemble and immobilize type I and type III collagen and generate stable gels and films, respectively, with the sufficient concentration of collagen required for in vitro binding assays, were developed. An optimized protocol for Eu(III) TRF of highly inert Eu(III)-DO3Aam-based complexes is presented. Finally, an optimized in vitro microplate Eu(III) TRF assay to measure the K_d of Eu(III)-labeled ligands toward immobilized type I and type III collagen is demonstrated (Figure 1).

Protocol

NOTE: All product information used for this work is presented in the Table of Materials. 1. Collagen immobilization NOTE: Ensure each well in the microplate used during the binding assay has adjacent wells free to avoid cross-fluorescence. Carry out this part of the protocol on ice because collagen self-assembles at rising temperatures and pH levels. Perform this procedure in a tissue culture hood and under sterile conditions because …

Representative Results

Assessing the stability and concentration of type I and type III collagen immobilized in gels/films The quantification of the collagen concentration immobilized per well was carried out using three different conditions: a) in wells without washing with PBS after immobilizing the proteins (no wash); b) in wells with a wash step (twice with PBS) after immobilization to remove any uncoated protein; c) in wells after incubation with PBS for 1 h (PBS mimic experiment). The PBS incubation mimicking step …

Discussion

This work presents a reproducible method for immobilizing type I and type III collagen. It also demonstrates a protocol for acquiring, analyzing, and interpreting in vitro Eu(III) TRF binding data to characterize the binding properties of a candidate ligand toward type I and III collagen. The protocols for immobilizing type I and type III collagen presented here were developed and optimized considering previously published work on type I and type III collagen fibrillogenesis in vitro^13…

Materials

10x PBS	Gibco	14200075	Use this to make 1x PBS by diluting in water (1:10)
2M HCL			Made in house and details are in the supporting document
2M Sodium hydroxide +2M Glycine			Made in house and details are in the supporting document
Cell-star 96 well  microplate	Greiner Bio-One	655 160
DELFIA enhacement solution	Perkin Elmer	1244-104
Ice
Infinite 200 PRO NanoQuant microplate reader	TECAN
Non-binding (NBS) 96 well microplates	Corning	3641
pH electrode Inlab Routine	Mettler Toledo	51343050
pH meter (sevenCompact)	Mettler Toledo
Pierce BCA protein assay kit	Thermofisher	23227
Tissue culture incubator (37 °C, 5% CO2)
Type I bovine collagen, 3 mg/mL	Corning	354231
Type III human placenta collagen, 0.99 mg/mL	Advanced Biomatrix	5021