Endogenous and Exogenous Cross-Linking

 Written by: Ayush Jain, Seth Liyanapathirana, Daniel Xie

Learning Objectives

This Exemplar will teach the following concepts from the ACS Examinations Institute General Chemistry ACCM:

II. A. 2. a. Bonds formed between two nonmetals tend to be covalent, while bonds formed between a metal and a nonmetal tend to be ionic in character. 

II. C. 1. a. Not all atoms combine to form stable bonds, but when a bond forms it is always accompanied by a lowering of the energy. 

III. F. 1. b. An important reason to define functional groups is that they allow the prediction of reactivity patterns.

VII. C. 2. e. Species that appear in a reaction mechanism but not in the overall reactions are called intermediates and their identification can provide important evidence about how well the mechanism describes the reaction.

VII. E. 2. a. Catalysts provide an alternative reaction pathway that lowers this activation energy.

 

Introduction: What is Cross-Linking? 

Cross-Linking is the process in which proteins are manipulated to cause the formation of covalent bonds. There are two main categories of cross-linking, Endogenous (naturally occurring) and Exogenous (artificially occurring), and we will be analyzing both to get a better understanding of cross-linking as a whole.  

 

Lysyl Oxidase-Mediated Cross-Linking (Catalyst)  

Lysyl Oxidase-Mediated Cross-Linking is a form of endogenous cross-linking. This cross-linking occurs naturally throughout our bodies.

 

 Definition: Endogenous Cross-Linking

Endogenous cross-linking refers to the natural, physiological cross-linking that could occur more commonly with age or in patients with diabetes.¹ This chemical process could explain why keratoconus cases are often worse in patients in their adolescence or early adulthood, whereas more middle-aged and beyond patients tend to have more stability due to the body more naturally creating this cross-linking reaction.² (more on this specific disease later)

 

Generally, when more Lysyl Oxidase is available, more cross-linking is witnessed because Lysyl Oxidase is enzymatic (serves as a biological catalyst to the cross-linking reaction). The reaction, resulting in the formation of covalent bonds, is shown in Figure 1 below. We can see the intermediate reacting with the aldehyde if we follow the path from A to B. If we follow the path from A to C, we can see the intermediate reacting with a lysine, then regardless of if it takes the path of A to B or A to C, it leads to D, where the cross-linking covalent bonds form.³ 

 

Figure 1. Cross Linking Explained.³

 

Definition: Enzyme

Enzymes are biological catalysts for biochemical reactions. Catalysts serve the purpose of speeding up a reaction by lowering its activation energy.

It is still debated in the literature if this form of cross-linking is age-related. New research suggests that the lysine glycation in collagen increases with age and not cross-linking (see Further Reading for more).⁴ 

 

Practice Problem # \(\PageIndex{1}\)

What Functional Groups are present in the amino acid Lysine (image below is from pubchem.ncbi.nlm.nih.gov)

 

Click Here for Solution

There are just two different functional groups present an amine (NH₂) and a Carboxyl (COOH). 

Amino Acids

Lysine falls in the group of amino acids. Amino acids are characterized by having these same two functional groups. 

 

Advanced Glycation End (AGE) Product-Mediated Cross-Linking (No Catalyst)

Advanced Glycated End (AGE) Product-Mediated Cross-Linking is the second form of endogenous cross-linking. Once again, this means that this form of cross-linking occurs naturally. However, the main difference between this and Lysyl Oxidase-Mediated Crosslinking is that this is non-enzymatic, meaning it does not have a catalyst. This form of cross-linking is seen more in patients with diabetes mellitus (DM) because having the condition relates to a surplus of sugar or glucose which opens up the possibility for the Maillard Reaction.³

 

Definition: Maillard Reaction

Found in 1912 by Louis-Camille Maillard to describe the reaction of proteins and sugars without an enzyme (non-enzymatic glycation).³ This is a rather complex reaction, and we refer to the end products as AGE products (Advanced Glycation End products). One of these products at the end of the Maillard Reaction is the cross-linking of collagen and other proteins.

Where else is this reaction found?

The Maillard reaction is also found in food! Generally, it describes amino acids and sugars reacting. The reaction occurs in browning food and gives it its distinct flavor.³ It is seen in almost every baking process today.⁵

 

Looking at Figure 2, part A shows this reaction between the protein and the excess sugar (glucose). Then, as this reaction continues till C, we see that one of AGE products is the collagen crosslinks.³

 

Figure 2. AGE Product-Mediated Cross-Linking.³

 

Corneal Cross-Linking Photochemically with Riboflavin and UV-A Light

Now that we have discussed how cross-linking can naturally occur, we can discuss how scientists have manipulated this process artificially through a technique known as exogenous cross-linking. The most common exogenous procedure is known as Corneal Crosslinking using Riboflavin and UV-A light.

 

Definition: Exogenous Cross-Linking

Creating the collagen cross-linking artificially via a procedure is the general idea behind exogenous cross-linking.¹

 

The premise of the corneal cross-linking procedure is to create a photochemical reaction (reactions induced by energy in the form of light) that forms covalent bonds both between two collagen molecules and between the collagen molecules and the proteoglycans. Once the riboflavin drops are added to the eye, as seen in Figure 3 part B, and light is added, as seen in Figure 3 part C, the riboflavin generates reactive oxygen species, which induce the covalent bonds to occur.⁶  D in Figure 3 is the final product of the procedure. We can directly compare this image to A to see the cross-linking forming in the cornea. 370 nm UV-A light was chosen as the wavelength because riboflavin was noted to have a peak absorption at this wavelength.⁶ The amount of energy carried by the wavelength can be calculated via Planck’s law in the practice problem below. Using the wavelength of maximum absorbance means that most (ideally all) of this energy is transferred to the reaction occurring in the eye.

 

Figure 3. Cross-Linking Explained ⁷

 

Practice Problem # \(\PageIndex{2}\)

How much energy (J) does 370 nm of UV-A light providing in the cross-linking reaction?

Key Equations & Constants

Planck's Law

E = hv = hc/(λ)

E = Energy (J)

h (Planck's Constant) = 6.626 • 10^-34 J • s

c (Speed of Light) = 2.99 • 10⁸ m/s

v = frequency (Hz)

λ = wavelength (m)

Click Here for Solution

E = hc/(λ)

E = (6.626 • 10^-34 J•s) x (2.99 • 10⁸ m/s) / (3.7• 10^-7 m)

E = 5.369E-19 J per photon

 

Additionally, we can further analyze the general reaction that results in the forming of covalent bonds between the collagen molecules in Figure 4. As seen at the start of the reaction (A in Figure 4), UV-A light and riboflavin are added. As mentioned earlier, researchers chose a wavelength of 370 nm for this procedure because this is the max absorption wavelength for riboflavin, meaning ideally, we will have 5.369E-19 J per photon absorbed to the eye (calculated in the problem above). The energy absorbed from the UV-A light allows free radicals to be generated on the collagen and the proteoglycans.³ Then, as we move to the last part of the reaction (B in Figure 4), we get our cross-linking reaction and the formation of our desired covalent bonds through a reactive nucleophile (like OH- group) reacting with the intermediate (imidazolone). 

 

Figure 4. Corneal Cross-Linking using Riboflavin.³

  

This form of cross-linking is used to cure diseases like Keratoconus in the eye.

Definition: Keratoconus

Keratoconus is an ophthalmic disease that causes the thinning and ectasia of the cornea in the eye and leads to vision impairment and other problems regarding eyesight. Ectasia occurs when the cornea becomes weak and distorts.⁸ Ectasia occurs from a lack of connections between collagen fibrils and the proteoglycan matrix.⁸ The proteoglycan matrix is composed of proteins found in the connective tissues that increase support and structure within the body and its tissues.⁸ In Figure 5, we can see how the cornea distorts and exhibits ectasia. Collagen fibrils are necessary for cell attachment to macromolecules such as the proteoglycan matrix. Because Keratoconus causes the weakening of the cornea, corneal-cross linking is used to rebuild and strengthen the tissues in the cornea, thus improving eye function and structure.

 

                           

Figure 5. Keratoconus Illustrated ⁹

 

Parallels Between The 3 Cross-Linkings

Even though the mechanisms for the different forms of cross-linking are different, they all result in the product of covalent bonds between two nonmetal elements of two collagen molecules. In all three cross-linking forms we explored, we see how the different functional groups react with intermediates to result in collagen cross-linking. Another parallel between the three is that all of them form stable bonds. When stable bonds form, the overall energy is lowered.  

References

1. Blackburn BJ.; Rollins AM.; Dupps, Jr. WJ. Biomechanics of Ophthalmic Crosslinking. Transl Vis Sci Technol. 2021, 10(5): 8. doi: 10.1167/tvst.10.5.8. 

2. Ashwin PT.; McDonnell PJ. Collagen cross-linkage: a comprehensive review and directions for future research. BR J Ophthalmol. 2010, 94(8): 965-70. doi: 10.1136/bjo.2009.164228. 

3. McKay TB.; Priyadarsini S.; Karamichos D. Mechanisms of Collagen Crosslinking in Diabetes and Kerataconus. Cells 2019; 8(10): 1239. doi: 10.3390/cells8101239.

4. Haneef A.; Giridhara Gopalan RO.; Rajendran DT.; Nunes J.; Kuppamuthu D.; Radhakrishnan N.; Young TH.; Hsieh HY.; Prajna NV.; Willoughby CE.; Williams R. Chemical Cross-Linking of Corneal Tissue to Reduce Progression of Loss of Sight in Patients With Keratoconus. Transl Vis Sci Technol. 2021, 10(5), 6. doi: 10.1167/tvst.10.5.6

5. Tamanna N; Mahmood N. Food Processing and Maillard Reaction Products: Effect on Human Health and Nutrition. Int J Food Sci. 2015: 526762. doi. 10.115/2015/526762.

6. Sorkin N.; Varssano D. Corneal Crosslinking: A Systematic Review. OPH. 2014, 232(1): 10-27. doi:10.1159/000357979. 

7. Maier P; Reinhard T; and Kohlhaas M. Corneal Collagen Cross-Linking in the Stabilization of Keratoconus. Dtsch Arztebl Int. 2019, 116(11): 184–190. doi: 10.3238/arztebl.2019.0184. 

8. Stammers M.; Ivanova IM.; Niewczas IS.; Segonds-Pichon A.; Streeter M.; Spiegel DA.; Clark J. Age-related changes in the physical properties, cross-linking, and glycation of collagen from mouse tail tendon. J Biol Chem. 295(31): 10562-10571. doi: 10.1074/jbc.RA119.011031.

9. Keratoconus. https://www.mayoclinic.org/diseases-conditions/keratoconus/symptoms-causes/syc-20351352 (accessed 2022-12-06)

Further Reading 

1. More on Photochemistry: http://photobiology.info/Photochem.html

2. More on debate on Age-related Changes on cross-linking: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7397091/

3. More general information on Keratoconus: https://www.mayoclinic.org/diseases-conditions/keratoconus/symptoms-causes/syc-20351352