Titin expression in human articular cartilage and cultured chondrocytes: A novel component in articular cartilage biomechanical sensing?
(Antibody Recognition and RT-PCR applications...)
The objective of this experiment was to test for the presence of titin on human articular cartilage tissue (e.g. joints). This large protein has previously been detected on heart and skeletal muscle tissue, where a biomechanical strain-bearing role has been attributed to it. "...Titin accounts for most of the passive elastic response retention that prevents over stretching... acts as an adjustable spring element..." Being this the case, it might be reasonable to look for the expression of this protein on other stress/strain-bearing tissues where elasticity is also required (e.g. joints).
Cartilage samples were taken from three groups of donors: Adults with osteoarthritis (OA), adults without OA, and infants (disgusting sampling). Immunolabelling was done with specific antibodies for "four" different domains of Titin; the N-terminal Z1-Z2 domain, the Novex III exon, the PEVK region, and N2A region (Fig.1). [I have to point out that even when only "three"antibodies were mentioned in the abstract, the paper showed results for "four" antibodies. Even section 2.3 (immunohystochemistry) doesn't make it clear (maybe it is me... I'm open to suggestions)].
However, it seems interesting to see positive results for titin presence in cartilage tissue (previously detected on muscle only). More than just existing on cartilage tissue, titin differences between Osteoarthritis and normal tissues were not detectable (Fig. 2,3). This suggests a crucial structural role of titin in cartilage.
Another result that called my attention was the detection of titin in extracellular matrix (ECM), when it was claimed to be intracellular (Fig. 2-4). This has two possible explanations:1) "a cross-reaction with other member of the Ig-domain gene superfamily, to which titin belongs" (detection of something else that has a domain in common with titin), and 2) titin has both intra- and extracellular strain-bearing functions.
Third interesting outcome: Some of the infant samples showed negative results for N2A region of titin (table 1, fourth column). This was associated to possible expression of a different titin isoform during maturation of the cartilage. This changes can be related to skeletal growth or mechanical load needs. This is a good point in favor of the specificity of antibody recognition (optimum trial and error, huh?). There were also discrepancies on the OA patients results (table 1, columns 2 & 3). This was similar to findings of aberrant forms of titin on patients with OA or Charcot Marie Tooth disease (Banes et. al. 2004). This is open to argument, though. Banes' aberrations where on the N2A region while aberrations here might be on Z1-Z2 (X112 113 X) and PEVK (9D10 ) regions.
Presence of titin was also shown by RT-PCR done from cells cultured from the samples. However, some domain fragments could not be amplified (see table2, Fig. 5). Again, might be related to the titin isoforms during cartilage maturation. Howeer, PEVK region was detected in all tissues by antibody recognition but not by RT-PCR. This case remains not clear. Further research will be required to clarify this.
Secondary Reference Sites:
1- http://www.ks.uiuc.edu/~ericlee/Telethonin/
2- http://www.psc.edu/science/2000/schulten/rude_mechanicals.html
Cartilage samples were taken from three groups of donors: Adults with osteoarthritis (OA), adults without OA, and infants (disgusting sampling). Immunolabelling was done with specific antibodies for "four" different domains of Titin; the N-terminal Z1-Z2 domain, the Novex III exon, the PEVK region, and N2A region (Fig.1). [I have to point out that even when only "three"antibodies were mentioned in the abstract, the paper showed results for "four" antibodies. Even section 2.3 (immunohystochemistry) doesn't make it clear (maybe it is me... I'm open to suggestions)].
However, it seems interesting to see positive results for titin presence in cartilage tissue (previously detected on muscle only). More than just existing on cartilage tissue, titin differences between Osteoarthritis and normal tissues were not detectable (Fig. 2,3). This suggests a crucial structural role of titin in cartilage.Another result that called my attention was the detection of titin in extracellular matrix (ECM), when it was claimed to be intracellular (Fig. 2-4). This has two possible explanations:1) "a cross-reaction with other member of the Ig-domain gene superfamily, to which titin belongs" (detection of something else that has a domain in common with titin), and 2) titin has both intra- and extracellular strain-bearing functions.
Third interesting outcome: Some of the infant samples showed negative results for N2A region of titin (table 1, fourth column). This was associated to possible expression of a different titin isoform during maturation of the cartilage. This changes can be related to skeletal growth or mechanical load needs. This is a good point in favor of the specificity of antibody recognition (optimum trial and error, huh?). There were also discrepancies on the OA patients results (table 1, columns 2 & 3). This was similar to findings of aberrant forms of titin on patients with OA or Charcot Marie Tooth disease (Banes et. al. 2004). This is open to argument, though. Banes' aberrations where on the N2A region while aberrations here might be on Z1-Z2 (X112 113 X) and PEVK (9D10 ) regions.
Presence of titin was also shown by RT-PCR done from cells cultured from the samples. However, some domain fragments could not be amplified (see table2, Fig. 5). Again, might be related to the titin isoforms during cartilage maturation. Howeer, PEVK region was detected in all tissues by antibody recognition but not by RT-PCR. This case remains not clear. Further research will be required to clarify this.
Secondary Reference Sites:
1- http://www.ks.uiuc.edu/~ericlee/Telethonin/
2- http://www.psc.edu/science/2000/schulten/rude_mechanicals.html
5 comments:
Man! I think my posting looks awesome! BTW I totally agree with it!
2 out of 1 Vis agree: good post.
Great article V, and perfectly-timed as a follow-up article to last Friday's lecture on anti-body mechanics and Western blots. Besides the actual contents of the research, I notice that those Western blots look just stinking AWESOME. Perfect lines. No smears. Quite unlike my own. T.T
I'm still not quite sure what's going on though. The titin has 4 recognizable parts and there are 4 AB that can bind to them? If I look at those pictures, some AB stain the whole cartilage and others only stain cells. Does that mean the titin is some kind of ginormous string that has one domain inside of cells and other parts outside?
David answer:
Indeed the pictures show stained areas on the cartilage tissue, outside if the cells. This was actually one of the most remarkable findings of the paper. Titin had not been reported as an exracellular protein, and now it is found on extracellular matrix (ECM)! As they said, there are two theories for this:
1- They antibody recognized another protein that has a domain in common with titin
2- Titin is present on the ECM also -maybe has a strain-bearing role there too-
You are also right on the part of the four antibodies. They use four antibodies. Each of them recognizes a different region of the protein (which BTW is HUGE). This is good in a sense that each antibody recognition helps you to corroborate the results. In other words, if you get all four antibodies attached to "something" there is less doubt that you are recognizing the right subject.
Hey;I'd like to say here that this guys reported a "not detectable differences" when comparing OA and normal cartilage staining. However, they did not mentioned any quantitative analysis. Good vision, huh? If they were taking BioEng115, they would've done some "imageJ" on those pictures.
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