Molecular Basis of the Disease State

vEDS is a disease of Type III Collagen, which plays an essential role in the structural support and stabilization of a wide variety of structures, including the walls of arteries and veins. Vascular Ehlers Danlos can be caused by a wide variety of mutations (250+), including insertions, deletions, and missense and nonsense mutations in the COL3A1 gene (Byers et al, 1979). The COL3A1 gene is extremely large, and encompasses more than 50 exons (Germain and Herrera-Guzman, 2004). Many patients have more than one mutation, and many of these mutations are unique to one family, showing that vEDS has a strong genetic component. The COL3A1 gene is essential because it encodes alpha-chains, the precursors to Type III Collagen; Col3 is an essential part of the extra cellular matrix, and must be synthesized, it cannot be obtained from the diet. Due to the wide variety of mutations, vEDS can actually be triggered via several different mechanisms.

collagen
Model of a portion of the triple helix of Col3. All three helices are present in the figure (left, middle, and right), and each is made up of three alpha-chains (magenta, pink, and yellow) (Source: Boudko, Sergei P., Jürgen Engel, Kenji Okuyama, Kazunori Mizuno, Hans Peter Bächinger, and Maria A. Schumacher. “Crystal Structure of Human Type III Collagen Gly991-Gly1032 Cystine Knot-Containing Peptide Shows Both 7/2 and 10/3 Triple Helical Symmetries.” The Journal of Biological Chemistry 283, no. 47 (November 21, 2008): 32580–89. doi:10.1074/jbc.M805394200).

Structural Disruption

To form one Col3 protein, three monomeric alpha-chains bind together and twist around each other in a corkscrew-like shape to form one, triple-helical protein; macroscopic collagen is made up of many of these homotrimer Col3 units bound together. These triple helices can be seen in the figure above. Each individual alpha-chain is composed of many glycine-proline-hydroxyproline repeats (Boudko et al, 2008). Although not solely composed of these three amino acids, the repeats of proline and hydroxyproline are useful because both are relatively inflexible cyclic amino acids that give each Col3 chain strength and stability. Repeating patterns of these three amino acids are generally located on the outside of the helices, and thus are important for binding between alpha-chains, which is essential for stability of the triple helix in Col3. Mutations of the glycine in the Gly-Pro-HydroxyPro motif account for the large majority of vEDS mutations, and this is highly disruptive to the overall structure of Col3; glycine is extremely small, so the substitution for a larger and/or charged amino acid can cause increased steric or electronic hindrance. This can greatly destabilize the protein and potentially preventing the formation of the triple helix, making the collagen nonfunctional (Jorgenson et al, 2014). Even if the triple helix is stable enough to form, a mutation may disrupt the shape of the helix, which is problematic because every Col3 chain is bonded to another collagen chain; the specific shape and diameter of the collagen chain is essential to normal function, as chains that are too thick or too thin are dysfunctional or nonfunctional (Wu et al, 2010). A single mutation in the Gly-Pro-HydroxyPro motif, then, could decrease the stability of the entire triple helix, which could correspondingly decrease the strength and stability of the entire collagen polymer chain (Boudko et al, 2008). The collagen polymer becomes messy and disorganize

Collagen
Microscopy image of collagen fibers. The top of Fig A shows normal collagen, which is nicely aligned in long, thing fibrils. The Top of Fig B shows the collagen of a patient with vEDS. This collagen is highly irregular and disjointed, and therefore likely nonfunctional. (Source: Mao, Jau-Ren, and James Bristow. “The Ehlers-Danlos Syndrome: On beyond Collagens.” Journal of Clinical Investigation 107, no. 9 (May 1, 2001): 1063–69).

 

Although the three alpha chains are stabilized by inter-chain bonding between the three helices of Col3, the protein is also stabilized at the C-terminal end via a cysteine knot. This knot holds the triple helix together using three disulfide bonds between each alpha-chain, which provides significant stabilization of the entire structure. The most severe forms of vEDS have a mutation in the coding region for the C-Terminal cysteine knot, implying that this region plays an essential role in the stability and functionality of Col3 (Boudko et al, 2008). The Gly-Pro-HydroxyPro motif repeats in this knot region as well, and mutating the glycine residue has been shown to be particularly detrimental to collagen stability. Interestingly, even though this region is termed a “knot,” it is not a static structure; instead, it is highly mobile and can take several different conformations. This is useful because it allows the triple helix of Col3 to change shape, which is probably important during the polymerization of collagen into long chains. Unfortunately, these different shapes of the knot distribute electron density, allowing only a partial crystal structure to be obtained, as shown below (Boudko et al, 2008).

Collagen
Many collagen triple helices arranged and bound together, creating a much larger collagen fibril that can provide structural integrity to surrounding tissue (Source: Boudko, Sergei P., Jürgen Engel, Kenji Okuyama, Kazunori Mizuno, Hans Peter Bächinger, and Maria A. Schumacher. “Crystal Structure of Human Type III Collagen Gly991-Gly1032 Cystine Knot-Containing Peptide Shows Both 7/2 and 10/3 Triple Helical Symmetries.” The Journal of Biological Chemistry 283, no. 47 (November 21, 2008): 32580–89. doi:10.1074/jbc.M805394200).

 

 Post Translational Modifications

In addition to structural and stability considerations, it has been shown that mutated monomeric alpha-chains take longer to form the requisite trimer of Col3 (Mizuno et al, 2013). In this study, the glycine of the Gly-Pro-HydroxyPro repeat was mutated, which caused an observable delay in the formation of a collagen trimer. This is problematic because each monomer is being post-translationally modified (PTM) during trimerization. Normally residues that are crucial for bonding or stability are moved into the middle of the helix during the trimerization process, where they are shielded from modification; however, since trimerization takes significantly longer in vEDS patients, the alpha chains are exposed to the cellular milieu for a longer period of time, and thus are more likely to be modified, usually via the addition of glycosyl units. Glycosylation of some, select residues is normal, but the arbitrary addition of glycosyl units to many residues can drastically affect the shape of each alpha-chain, and prevent the trimerization into Col3. Glycosyl units are also highly reactive, and can indiscriminately bind to other macromolecules in the cell, further disrupting the normal structure of collagen.

Nonsense Mutations

In addition to these more structural changes, some vEDS patients have nonsense mutations, in which the mRNA for Col3 becomes shortened during transcription due to an early stop codon. The alpha-chains that are translated from this mRNA are also shortened, which shortens, or prevents the formation of, the resulting triple helix; in both instances, the Col3 is dysfunctional. If the triple helix does form, it has vastly decreased thermal stability, and can spontaneously dissociate into individual alpha-chains, even at normal body temperatures (Superti-Furga et al, 1988).

Some of these shortened mRNA chains may also automatically activate nonsense-mediated decay pathways, an RNA degradation mechanism. This pathway decreases the number of transcripts that code for alpha-chains; this is problematic because some vEDS patients are surprisingly actually able to produce some normal alpha chains. The disease is autosomal dominant, and collagen has three separate chains, so the laws of probability state that  7/8 of collagen will contain one or more mutated chains; one mutant chain is usually enough to make the entire Col3 unit dysfunctional; about 1/8 of the collagen being produced should be normal. However, in mutations that cause mRNA degradation, even less normal collagen is being produced, which only serves to worsen symptoms.

Haploinsufficiency

A few vEDS mutations have been shown to cause haploinsufficiency, which also decreases the amount of collagen being synthesized (Schwarze et al, 2001). In this case, one allele codes for normal collagen, while the other allele produces mutated collagen. vEDS is usually inherited autosomally dominant, but in about 5% of all patients, it can also be haploinsufficient. These patients produce 50% of normal collagen and 50% of mutated collagen; frequently the mutated collagen is destroyed via nonsense mediated decay before translation. These patients produce much more normal collagen than the typical vEDS patient, and thus manifest the disease later in life, and generally with more mild symptoms.

 Manifestations of the mutations

In all of the above mechanisms, the end result is the disruption of individual alpha chains, which is inherently problematic because it prevents the formation of normal triple helices. The triple helix is absolutely essential for normal Col3 function, as this is an extremely important part of the tertiary and quaternary structure of the protein. Without a norma triple helix, the collagen is simply nonfunctional, and cannot provide support for other tissues in the body. While collagen itself does not play a major metabolic role, it plays an absolutely essential part in providing structural support and tissue organization that allows the body to function as normal.

Since some normal triple helices are being produced by vEDS patients, symptoms often do not manifest for until the teenage years, or even beyond; however, functional Col3 is still essential for life, and it is only being incorporated into the walls of major blood vessels or organs at very small rates. Therefore, it is only a matter of time before the organs and vessels become so weak that they rupture, even when only exposed to a tiny amount of force. Thus, regardless of the specific mechanism of action, the symptoms of vEDS all stem from the disruption of the triple helix and general lack of bioavailability of functional Type III collagen.

Ehlers Danlos Syndrome, Vascular Type

History and Metabolic Context

Disease Treatment

Conclusions and Proposals for Future Work

6 Replies to “Molecular Basis of the Disease State”

  1. This was a really interesting read! I was particularly intrigued by the C-Terminal cysteine knot. You mention that, until recently, getting a structure of it has proven difficult because of how mobile it is. I was wondering if they found what the dominant conformations were, and more importantly, what the significance of those conformations is. Basically, I am curious as to why the conformation of the Col3 triple helix would change. Is it for how it assembles into larger collagen fibers or does it play a role in PTM? Or something else entirely?
    Sorry for so many questions. It just seems like a really neat structural element.

    1. Hi Matt,

      While there has been some success in obtaining that crystal structure, it has been somewhat minimal. The group was only able to get a partial structure, and even that was only about 40 amino acids in length. They were, though, able to find one structure for the knot, and showed that it had a lot of super-helical properties, including triple-helical symmetries. My understanding is that the changes in structure of the collagen knot are not extremely drastic, but are used to facilitate binding, both within the individual triple helix and with neighboring triple helices. The movement of the knot provides a more flexible conformation that permits the formation of various hydrogen and ionic bonds, which are essential to the stability, and thus the function, of the collagen polymers.

      I think it is important to note that this crystal structure was extremely difficult to obtain, and that the generated structure of the knot may be a combination of all of the possible conformations of the knot. Thus, I think it is difficult to have a really definitive answer until the entire protein has been crystallized, which will give insight into the three dimensionality of the overall chain. It may simply be that the protein is forced to cycle through different conformations as it assembles in an effort to minimize steric and electronic hindrance.

      The paper is here, but it is a little heavy on the crystallography data: http://www.ncbi.nlm.nih.gov/pubmed/18805790

  2. I’m interested in knowing more about the mechanism for the mRNA degradation pathway, as this is something I am not familiar with. From your writing it appears as though this pathway degrades both normal and mutated mRNA for the Col3 – is that correct? If so, that is very interesting – and I’d like to gain a better understanding of why this pathway is unable to differentiate between the two.

    I am also a bit confused by the inheritance pattern of vEDS. You noted that “a few vEDS mutations have been shown to cause haploinsufficiency” but also that the majority of cases of vEDS are inherited through an autosomal dominant pattern. Do the others not lead to a disease state through haploinsufficiency? And if not, how do they create a disease state in haploid individuals? Are there any cases of haplosufficient mutations in the Col3 gene?

    1. Hello,

      Thank you for your questions! I’m sorry if it was unclear, but only the shortened mRNA for Col3 is degraded, not the mRNA of normal length. The body has a built-in mechanism called nonsense mediated decay, which detects abnormally shortened mRNA due to the presence of a mutation that introduces a stop codon at an incorrect location, causing early termination of transcription. These transcripts can bind to an activate UPF proteins, which can detect abnormally short transcripts and activate the signal cascade that results in the actual destruction and recycling of the mRNA. If you want to learn more about nonsense-mediated decay, here is a nice review article: http://www.sciencedirect.com/science/article/pii/S0092867400805425

      You are correct that only some mutations cause haploinsufficiency (only ~5% of vEDS patients, which in itself is a small number). vEDS patients with haploinsufficiency thus only produce 50% of the typical amount of collagen because one copy of the gene functions normally while the other is mutated. The mutated version is generally a nonsense mutation, leading to nonsense-mediated decay. However, the other copy of the gene does produce functional Col3, which tends to result in milder symptoms, and a slower progression of the disease, in which symptoms do not appear until later in life. These patients actually have enough collagen that they are able to function relatively well until later in life, when the body becomes frailer and is generally unable to cope with the lack of collagen.

      If you are interested in learning more, check out this article on haploinsufficient vEDS patients: http://www.nature.com/gim/journal/v13/n8/full/gim2011118a.html

  3. Hi Mike! Great job with an interesting disease. I notice you say, when talking about the mutations that cause vEDS, that vEDS can be triggered by a variety of mechanisms. Does this mean that one could have a mutation in the Col3 gene and not present with the disease until a certain physiological condition occurs?

    On another note, this is just something I’m curious about. In your project, you’re talking about vascular Ehlers Danlos. From what I understand from your well-written page, most of the symptoms of vEDS are caused from issues that prevent the proper formation of collagen triple helices. Maybe I’m not quite understanding the terminology correctly, but can these mutations in the Gly-Pro-HydroxyPro motif and problems with PTMs and RNA degradation cause other forms of EDS? If not, why are they only symptomatic of vEDS? Sorry if this is unclear!

    1. Hi Gabbie,

      Thanks for your questions!

      I don’t think vEDS is necessarily triggered by a specific physiological condition, but it is more an accumulation of problems over time. The patient is producing abnormal collagen from birth, but the symptoms tend not to appear until late teens through middle age. It isn’t known exactly why some patients present earlier and some later, but it likely is tied to the specific mutation, of which there are several hundred. Some mutations cause haploinsufficiency, in which the patient produces a decreased amount of normal collagen; this results in a generally delayed onset of vEDS with much fewer symptoms. Other mutations may simply produce solely deformed collagen, which cannot form the normal triple helices and thus causes major problems for the body. This patient would likely start to experience symptoms much earlier in life, and would be at high risk for arterial or organ rupture. There isn’t a great explanation as to why young patients do not present with symptoms, but my understanding is that the younger body is simply composed of tissues that are more elastic and resilient, and thus are less likely to burst. Thus, pediatric patients are able to rely on other structural support mechanisms that prevent rupture of important organs. Older adults (even without vEDS) have organs that are simply weaker and not very stretchable—add a lack of Col3 and it’s a disaster. Thus, as the body slowly ages and becomes less adaptable, the lack of collagen becomes apparent, and manifests with vEDS symptoms.

      You are definitely right that vEDS affects the triple helices, which causes detrimental weaknesses in Col3. Interestingly, vEDS mutations specifically affect Type III collagen, while other forms of EDS create mutated forms of other types of collagen. There are 29 different types of collagen in the body, and EDS can effect any combination of them; Classic EDS, for example, has mutations in Type V collagen. Many of the other forms of EDS also cause mutations in the quaternary structure of collagen, which results in many shared symptoms between the different classifications of the disease. vEDS is the only form that specifically causes weaknesses in blood vessels and organs though.

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