liver disease, Uncategorized

Smooth muscle autoantibodies

I had an idea of at at home auto antibody test. Reading about auto antibodies changes my strategy totally for this at home product. How can our bodies product anti bodies against a protein that only recognizes a protein epitope when the protein is associated with other proteins? Smooth muscle proteins, and actin in particular, are something I know well. What better way to answer this paradox?

Auto antibodies against a protein polymer?

Autoantibodies against F-actin have been considered a component of autoimmune hepatitis [1] and other liver diseases. In this particular study the authors used ELISA plates with polymerized actin in the wells such that the native conformation was conserved. [1].

Muscle proteins are produced in thymic epithelial cells

More than 85% of all genes encoded in the genome, including many tissue-restricted self-antigen genes, are detectable in medullary thymic epithelial cells. (mTECs) [2]. TEC engage in promiscuous gene expression that is under the control of the autoimmune regulatory (Aire) gene. [2]. This review went on to discuss differential gene expression and cluster thereof in the thymic medulla versus cortex…An Internet image search reveals Aire partnering up with RNA polymerase, DNA topoisomerase, and the usual transcription factor partners. These images also include DNA coming off histones that have become a little less packed in chromatid. let’s look at an image from teh Aire video and a blow up of the vicinity of the aorta smooth muscle actin gene ACTA2. Most of the proteins in the immediate neighborhood are not vascular smooth muscle proteins except HTR7, one of the 5-hydroxytrypamine (serotonin) receptors. This protein is also expressed in the brain.

Figure 1 An image of a video explaining how Aire regulates promiscuous gene expression. The inset image in the upper right was obtained from the NCBI database.

This is the 21st century “not so” dogma. In humans, loss of AIRE function causes the autoimmune polyendocrine syndrome type-1 that is characterized by severe organ-specific autoimmunity: parathyroid chief cells, steroidogenic cells of the adrenal cortex, pancreatic β-cells, gastric parietal cells, skin melanocytes, hepatocytes, and so on. [2] Samson and coworkers looked a single cell mRNA transcripts in TEC. [2] While mRNA transcripts for proteins found only in the parathyroid gland, may have been found in the same mTEC, they stopped short of determining whether these expressed proteins acted together to perform parathyroid functions. Some earlier studies leave some room for doubt as to whether gene expression might be coordinated in other way(s).[3]

Making mini-muscles in the thymus?

Some of this was addressed back in 1979 when samples of normal human thymus of different ages (4-63 years old) were studied by immunofluorescence microscopy. [3] The authors were interested in myasthenia gravis, what we now know is caused by auto antibodies against the smooth and skeletal muscle acetylcholine receptor. These authors used immunohistochemistry with antibodies against smooth and skeletal muscle proteins to compare thymus glands from normal and myasthenia gravis patients. [3]. These authors claimed to detect “myloid” like cells that stained with antibody against striated but not smooth muscle myosin. They further claimed disorganized filaments that resembled those from denervated skeletal muscle.[3] It should be noted that human skeletal muscle actin ACTA1is on chromosome 1 and MYH1, the gene for skeletal myosin heavy chain, is on chromosome 17… along with many other myosin isoforms.

Figure 2 Actin monomers (globular, G-actin) are illustrated by round cyan or green balls. G-actin polymerizes to form F-actin. In striated muscle actin is held in anchors called Z-lines. We now know that Z-lines contain many proteins in addition to actin and myosin. Dense bodies are the smooth muscle equivalent of Z-lines.
  • Chicken gizzard smooth muscle myosin antibodies reacted with TEC but not “myloid” cells.
  • Chicken gizzard smooth muscle actin antibodies reacted with TEC and “myloid”cells.
  • Human striated muscle myosin antibodies reacted with “myoid” cells but not the TEC.

The authors claimed that myoid cells occurred in the rounded and elongated variety and that they were a normal constituent of all thymusglands investigated in their study. They also claimed something that looked like Z-lines that are not found in non striated muscle. This myosin may have been one or more of the non muscle myosin isoforms that we were not aware of back in 1978. Aggregates of promiscuously expressed proteins are not presented in their entirety, are they? Okay, these self proteins still have to be digested in order to present to developing T cells. It appears that they are not allowed to assemble in their tissue specific manner but rather are digested as soon as they come off the ribosome. This study leaves some room for doubt.

Developing T cells in the thymus are not selected with thin and thick filaments of actin and myosin in the thymus. Something else has to happen first.

Cathepsin digestion is a requisite for T cell selection

In order to be presented as an antigen by TEC or antigen presenting cells in the circulation, proteins must first be digested with Cathepsins. Somehow there must be a difference in proteolytic fragments presenting in the thymus by TEC and those presented by antigen presenting cells in the circulation.

  • Cathepsins L, S, C, F, H, B, X, K, V and W are related to the protease papain. [4] which cleaves after an arginine or lysine preceded by a hydrophobic unit (Ala, Val, Leu, Ile, Phe, Trp, Tyr) and not followed by a valine.
  • Cathepsins D and E are also found in lysosomes but are aspartic acid proteases related to pepsin.[4] Pepsin preferentially cleaves at Phe, Tyr, Trp and Leu in position P1 or P1′. Skipping nuances of pepsin specificity, this enzyme is quite good at chewing up hydrophobic regions of proteins that tend to fold in upon themselves or aggregate with hydrophobic regions of other proteins. We see these clusters of pepsin (>pH 2, Pn2) sites in Figure 3. These sites are less exposed to the solution when myosin is found to actin (Figure 4).

Just for the sake of this thought experiment, PeptideCutter is used to generate the hypothetical peptides of the alpha smooth muscle actin sequence from UniProt and paste it into PeptideCutter. For the sake of this thought experiment we will use pepsin > pH 2 as a surrogate for Cathepsins D and E and trypsin, which also cleaves at Arg (R) and Lys (K), as a surrogates for the other cathepsins.

Figure 3, alpha smooth muscle actin was completely digested in silico. Many of the fragments are either too large or too small to fit into the stereo typical class IMHC. The iMHC image is from oncohemakey.com

In this thought experiment we see that many products of this complete digestion, courtesy of Expasy’s Peptide Cutter, are either too small or too large to fit into a classical class I MHC heterodimer. Are some 8-9 amino acid fragments are pulled from the digestion process before they are reduced single amino acids? For the sake of argument, let us suppose that F-actin auto antibody production is initiated by an airway or gastrointestinal site where we find smooth muscle actin. Say this infection causes smooth muscle cells to lyse and release their contents. Circulating T cells will not see this mess until antigen presenting cells come along, clean up the mess by phagocytosis, digest the mess, and present the products to T cells that wander by looking for fragments of guilty pathogens. As we see in figure 1, actin from muscles come associated with many other proteins that lysosomes must digest before getting to all the consensus sites that they prefer.

The surface of actin “looks” different when myosin binds

Half of a resent publication used a unique method to map the binding site of the S1 head of myosin on filamentous actin. [6] These authors used X-rays to generate hydroxyl radicals from the hydrolysis of water. These hydroxyl radicals oxidized amino acid residues that they came in contact with. Sites on actin monomers in contact with other actin monomers are slow to react with hydroxyl radicals. Likewise, sites in contact with S1 also are less likely to react with hydroxyl radicals. They then digested the actin filaments overnight with trypsin. They determined the extent of oxidation of the peptides by mass spectrometry. [5].

Figure 3 (of this post) came from figures 1 and 2 of reference [5]. The bottom left contains the predicted tryptic (blue) and pepsin (pink) fragments from figure 1 (of this post).

Oztug also and coworkers also created actin mutants with stragetically placed cysteines to which fluorescent reporter probes were attached. [6] They concluded that myosin subfragment S1 creates structural changes in these flexible double stranded F-actin polymers. S1 binding to F-actin reduced oxidation of all fragments, not just the ones directly interacting with myosin S1. One would think that the actin “breathing” they referred to might be further restricted if the S1 mysosin heads had remained attached to the filaments shown in Figure 2. While the authors only concerned themselves with actin surfaces exposed to hydroxyl radicals, we can anticipate changes in accessibility to cathepsin proteases in the lysosome also apply. This presents an added challenge to the TEC promiscuous protein.

Generating self reacting actin antibodies is easy

In 1983 it was shown that rabbits immunized with chicken gizzard and ascaris actin without any modifications would produce antibodies that recognized rabbit actin isoforms. [6]

Figure 5. From reference [6] Chicken gizzard F-actin was used to immunize rabbits. These antibodies recognized the non muscle actin in (a) mouse 3T3 fibroblasts, (b) rabbit stomach, gamma smooth muscle actin, (c) rabbit skeletal muscle alpha sk actin, and (d) rabbit kidney.

It is doubtful that Nishioka and and coworkers truly recognized the bizarre nature of their finding. [6] What sorts of auto antibodies would they have found if they had immunized their rabbits with thin and thick filaments from chicken gizzard with the proteins shown n Figure 2?

How might actin auto antibodies be generated?

  1. In TEC actin is mostly monomeric and rarely associated with myosin. The stretch of amino acids 340-360 is easily degraded by pepsin like cathepsins. These peptides are too small to be mounted in MHC complexes. T cells with receptors that can bind to this region escape the thymus.
  2. When muscle cells are injured, they release filaments of actin and myosin (Figure 2) into the circulation. Macrophage remove the debris by phagocytosis. The phagosomes fuse with lysosomes for digestion of the debris.
  3. Digestion of regions of actin bound to myosin is a bit slow until the myosin is digested to a point that it no longer binds actin. Some of these partially digested peptides become bound to MHC and presented to circulating T cells.
  4. The body produces antibodies against this region of F-actin that likes to bind myosin… but does not bind myosin at every single site on an F actin filament.

References

  1. Granito, A., Muratori, L., Muratori, P., Pappas, G., Guidi, M., Cassani, F., Volta, U., Ferri, A., Lenzi, M., & Bianchi, F. B. (2006). Antibodies to filamentous actin (F-actin) in type 1 autoimmune hepatitis. Journal of clinical pathology, 59(3), 280–284. https://doi.org/10.1136/jcp.2005.027367
  2. Sansom S. N., Shikama-Dorn N., Zhanybekova S., et al. 2014. Population and single-cell genomics reveal the Aire dependency, relief from Polycomb silencing, and distribution of self-antigen expression in thymic epithelia. Genome Res. 24:1918. [PMC free article]
  3. Drenckhahn D, von Gaudecker B, Müller-Hermelink HK, Unsicker K, Gröschel-Stewart U. Myosin and actin containing cells in the human postnatal thymus. Ultrastructural and immunohistochemical findings in normal thymus and in myasthenia gravis. Virchows Arch B Cell Pathol Incl Mol Pathol. 1979 Dec;32(1):33-45.
  4. Colbert JD, Matthews SP, Miller G, Watts C. Diverse regulatory roles for lysosomal proteases in the immune response. Eur J Immunol. 2009 Nov;39(11):2955-65 Cross Ref
  5. Oztug Durer, Z. A., Kamal, J. K., Benchaar, S., Chance, M. R., & Reisler, E. (2011). Myosin binding surface on actin probed by hydroxyl radical footprinting and site-directed labels. Journal of molecular biology, 414(2), 204–216. Free PMC article
  6. Nishioka, M et al. “Rabbit autoantibodies to actin induced by immunization with heterologous actins; a possible mechanism of smooth muscle antibody production.” Clinical and experimental immunology vol. 53,1 (1983): 159-64. Free PMC article

Leave a Reply