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Review Paper
Human Autoantibodies Reveal Titan as a Chromosomal Protein

Cristina Machado, Claudio E. Sunkel, and Deborah J. Andrew

Summary
    Autoimmune diseases are identified through several autoantibodies that react with epitopes on the nucleus, cytoplasm, and cell surface.  Autoantibodies can be used as a probe for the identification of new proteins and their genes, and human autoimmune sera have been used to identify a number of nuclear antigens.   Scleroderma is an autoimmune disease affecting many types of connective tissue with an unknown etiology, causing vascular lesions and tissue fibrosis.  Autoantibody targets in scleroderma are extremely specific, and although they typically bind to nucloelar antigens, some scleroderma autoantibodies react with metaphase chromosomes and the centrosome.
     Scleroderma serum was used to isolate a Drosphila melanogaster  gene through its binding to an epitope on mitotic chromosome in both human culture cells and Drosophila embryos.  This serum was then used to isolate a Drosophila gene, D-Titin,  encoding a chromosomal protein believed to be the homologue for vertebrate titin. Titin is a sarcomeric protein involved in muscle elasticity and myofibral scaffolding.  D-Titin  is expressed in striated muscle tissue, and two antibodies against D-Titin  localized it to the Z-disks of Drosophila  sarcomeres.  The D-Titin  also stained condensed mitotic human and Drosophila  chromosomes. Immunofluorsence with monoclonal and polyclonal antibodies proved that D-Titin  is localized to condensed mitotic chromosomes, and suggests that titin may also play a role in the structure of mitotic chromosomes.
    Titin is a large protein, between 2,993 to 3,700 kD, spanning half the sarcomere from the Z-disk to the M-line, a length of 1.2 mm in relaxed skeletal muscle.  Almost 90% of its weight is composed of Ig-like and fibronectin type-3 (FN3)- like repeats disbursed throughout the protein.  In addition the I-band area of vertebrate titin is composed of an area rich in proline, glutamic acid, valine, and lysine (PEVK domain).  This protein has many functions, and may act as a building block for myofibril assembly.  In addition, titin mRNA and proteins are expressed very early in the development of the sarcomere, and are seen in several regions of the sarcomere. The authors were interested to see what role titin played in  chromosome condensation during mitosis.
Results
 To identify the novel gene in Drosophila, human autoimmune sera were screened for nuclear components with cell cycle dependent distribution. Serum from patients suffering from scleroderma was studied, and analyzed to see if it would yield chromosomal staining in both human epithelial HEp-2 cells and Drosophila 0-2h embryos.  Figure 1A shows that both the human HEp-2 cells and Drosophila embryos showed low-level staining in prophase, with the chromosomes beginning to condense by interphase.  By metaphase there was a clear co-localization of serum and DNA, as the chromosomes condensed and separated.  In both anaphase and telophase, the serum and DNA are co-localizing as the chromosomes condense and separate.  This is showing that the chromosomal protein in the human autoimmune serum is associating with the chromosomes during mitosis.  Figure 1B uses a-LG (an antibody that recognizes the largest clone from titin) and propidium iodide (to detect DNA) to show the localization of DNA and titin during the metaphase and anaphase stages of mitosis in Hep-2 cells and Drosophila embryos.  Since these panels show the same pattern of migration as the serum and DNA from Fig. 1A, this proves that a-LG is localizing with the DNA during metaphase and anaphase.  Figure 1C uses a-KC (an antibody that recognizes the 5 most region of cDNA for titin), and propidium oxide (to detect DNA), to test for the staining of chromosomes during mitosis.  This panel showed the same staining pattern as the human autoimmune serum and a-LG, showing chromosome staining during metaphase and anaphase.  In all panels, Hep-2 cells are used as a positive control, to show chromosome condensation and migration during mitosis.  Figure 1 proves that titin is associating with the chromosomes during metaphase and anaphase.
 
Figure 2A shows the restriction map of the D-TITIN genomic region, from which the clones for D-Titin were generated.  Only one clone, phage clone 5, was isolated directly using the LG genomic DNA expression clone as a probe.  The other clones were isolated using DNA flanking from nearby P-elements, V(3)ET1 and V(3)ET2.  This restriction map is used to show the position of the different cDNAs.  The KZ cDNA encodes the NH2 terminus, and encodes an open reading frame (ORF) of 882 amino acids.  The NB cDNA has a 1kb ORF, and a 3 splice acceptor site and a 5 splice donor site.  The JT cDNA is a small ORF located from the Tamkum library.  Although they were unable to connect the genomic DNA from phage clone 5 to the adjacent clones, all the cDNA clones co-localized to the same region on a chromosome, cytological region 62C1-2, known to only encode one gene.  The author also could have attempted primer walking for the region between phage clone 5 and the next clone to try to join these sequences.  I also believe that the author should have shown the results of the in situ hybridization to polytene chromosomes, to prove that these regions do indeed all localize to one area.   Figure 2B shows a protein sequence alignment for the ORFs of the two D-Titin cDNAs (KZ and NB), chicken skeletal titin, and human cardiac titin.   For KZ cDNA there is a 28.6% identity and 58.3% similarity between D-Titin and chicken skeletal titin, and a 27.4% identity and 56.8% similarity to human cardiac titin.  For NB cDNA there is an 18.4% identity/48.9% similarity to chicken titin, and a 17.2% identity/47.7% similarity to human titin.  Although there does appear to be a high level of conservation, there is an unknown sequence of D-Titin, making protein sequence analysis troublesome.  Since the authors do not even know how long this sequence is, they cannot know if this sequence would drastically lower the conservation rate.  Figure 2C shows the sequences of the PEVK-rich ORF of human scleroderma autoimmune serum (LG) and the largest isolated cDNA (JT).  Both the LG gDNA and JT cDNA show a similar percentage of PEVK to vertebrate titin: 63% of PEVK in LG and 56.4% in JT, compared to 70% PEVK in vertebrate titin.  My problem is that the ORF is much smaller in JT cDNA, so the percentage of PEVK could be flawed since it will have a higher percent through its smaller size.  Also the authors do not say if NB cDNA and KZ cDNA also have similar percentages of PEVK in their ORFs.

Figure 3 is used to see if the gene that encodes the form of chromosomal titin is also the gene for the muscle form of titin.  Fluorescent in situ hybridization was used to detect RNA and protein of D-Titin in different developmental stages.  This figure shows that D-Titin is expressed early in somatic and visceral muscle cells in stage 11.  Both RNA and protein for D-Titin are seen in each developmental stage in all visceral and somatic muscles except for cardiac muscle.  This figure proves that the same gene encoding the titin that is associated with chromosome condensation in mitosis, is also present in developing visceral and somatic muscle cells.  The only problem that I see with this figure is that there is no vertebrate muscle for comparison, to test and see if D-Titin associates with the same types of muscle cells in vertebrates.

Figure 4 shows fluorescently labeled regions of a sarcomere of Drosophila adult thoracic myofibrils and larval gut muscle to test if the protein localized to a specific region of the sarcomere.  Figures 4A-F study the staining of adult thoracic myofibrils with different antibodies.  Figure 4A shows that a-KZ stains the Z-disks of sarcomeres, through comparison with the upper panel showing staining of Z-disks in thoracic muscle.  Figure 4B shows that a-KZ binds to the Z-disks, with the red palloidan binding to the filamentous actin of I-band region.  Figure 4C shows that binding of the human autoimmune sera to the M-line and Z-band staining with a-KC.  Figure 4D shows staining with a-KC and a-LG, showing binding to the Z-disk with a-KC, but while they appear to bind to a similar region there is no overlap.  Figure 4E staining with a-KC, bound to the Z-disk, and MIR which binds to the I-band near the I/A band region without a region of overlap.  Figure 4F shows staining with a-KZ and the anti Zr5/Zr6 polyclonal serum (binds to a-actinin), with an overlap of signals, presumably because of insufficient resolution in the microscope.  Figure 4G shows staining of third instar larval gut muscle with a-KZ.  This staining overlaps the phase-dark regions of the Z-disks in larval gut muscle (data not shown).
My problem with this figure is that in Figure 4G the author should have shown the staining of phase-dark regions for comparison with the a-KZ.  This is also the only example of embryonic tissue, and I wonder why he did not test embryonic tissue with the other antibodies.  Also while he does not provide any figures for the staining with pre-immune sera from a-KZ and a-LG, though he says that a-KZ does not stain while a-LG does.  The authors should show this, if only for a negative control.  Also the authors should have provided a positive control showing staining patterns for the different regions of the sarcomeres, instead he only shows what the Z-disk should look like.  They should also have tested for cross-reactivity with other antibodies, since the scleroderma serum bound to the whole length of the chromosome.  This does prove, however, that both a-KZ and a-LG bind to the Z-disk region of the sarcomere.
 
Figure 5 is an immunoblot of D-TITIN in three different types of cells, 8-24h embryos (after myogenesis), 0-2h embryos (before myogenesis), and HeLa cells.  This was used to test the size of the protein to see if it fell within the range known for titin proteins (2,993 to 3,700 kD).  Figure 5A tests for D-TITIN after myogenesis, when muscle cells are present.  There is a band present when the immunoblots were incubated with a-KZ (lane 2) and a-LG (lane 4) that appears to be the right size, but there is no molecular marker for a protein this large.  Since there is no band for either a-KZ or a-LG, this protein does not appear to be cross-reacting.  Figure 5B tests for the size of D-TITIN in non-muscle cells (embryos before myogenesis occurs).  Bands appear when the gel was incubated with a-KZ (lane 2) and a-LG (lane 4), showing that D-TITIN is present, and it also appears to be the same size as D-TITIN in muscle cells through. In addition, like muscle cells, pre-immune sera from a-KZ and a-LG do not show any bands.  Figure 5C shows the expression of protein extracts in HeLa cells after they were separated by SDS-PAGE.  These cells show the same banding patterns as both muscle and non-muscle cells of Drosophila embryos, proving that it is present in the right size and not cross-reacting.  The only problem that I have with this figure is that there is no molecular marker showing that the band identified is actually within the size range for a titin protein, since the largest marker they have is 584kD, and titins are normally 2,993 to 3,700 kD.
 
Figure 6A tests if antibodies to vertebrate titin would stain Hep-2 cells.  Eight antibodies were used, but only the five that showed strong staining were shown in Figure 6A.  Mouse monoclonal antibodies BD6 and CE12 recognize epitopes on the A-band, and both showed strong staining of condensed chromosomes.  The mouse monoclonal antibody 9E10 that recognizes the PEVK domain of the I-band was not shown in Figure 6A, because it gave weak staining.  Two rabbit monoclonal antibodies, N2A that recognizes the I-band epitope bound weakly, while A168 that recognizes the M-line epitope showed strong staining.  The MIR human autoimmune serum that bound to the I/A- band epitope showed strong staining of condensed chromosomes and the mitotic apparatus.  N2A, MIR, BD6, CE12, and A168 showed staining indistinguishable from that seen in the original scleroderma serum and antibodies to D-TITIN protein.  Two vertebrate antibodies, anti-Zr5/Zr6 and T12,  did not bind to the Hep-2 cells, because they are associated with the NH2-terminus region of titin.  The authors conclude that while titin binds to both human cells and Drosophila embryos, its muscle and chromosomal forms may vary in their NH2 terminus.  My only problem with this figure is that the authors did not include antibodies that did bind to the chromosomes, and included N2A which stained poorly, yet left out 9D10 which also stained poorly.  It would have been useful to have another signal that showed weak staining for comparison of the strength of other signals.  Figure 6B is a diagram of the sarcomere, showing the location of the epitopes for each antibody, and the distribution of the different bands of the sarcomere.
Conclusions/Discussion
     D-Titin was the first titin autoantibodies to be identified in scleroderma serum, the first time titin was identified as a chromosomal component, and the first Drosophila gene to be cloned from human autoimmune serum.
     Titan is uniformly distributed along mitotic chromosomes, and may play a part in their condensation, perhaps similar to protein scaffolding in sarcomeres.
    Titan may act as a "molecular ruler" determining the axial diameter and/or length of condensed mitotic chromosomes.
    Titan because of its elasticity may also play a role in preventing chromosomal damage and breakage during mitosis.
    Muscle and chromosomal versions of titin may vary in their NH2 termini, perhaps because of alternate splicing
    Titin is involved in protein scaffolding, like topoisomerase II and the SMC proteins, but was not detected in previous experiments because of its large size, since previous 12.5% gels did not test proteins over 200kD and titan weighs ~3000kD.
     Titin may act as a new autoantigen in scleroderma, since it was isolated from scleroderma autoimmune serum.
Future Experiments
     Check to see if titin binds to interphase chromosomes.  Since the author had indicated that a higher rate of binding occurs in some areas, determine what those areas are, and see if they relate to areas on the chromosome where titin is more active.
     Use a better gel (the author recommends 2.5%-7.5% gradient gels) to determine not only the weight of D-TITIN, but also if there is a difference between chromosomal and muscle titin.  If there is a difference try to sequence the NH2 terminus and look for differences that could indicate an alternate splice site.
     Probe D-TITIN with the other titin genes of the Drosophila family and see if they are on the same gene.  Even if they are on different genes, use a BLAST query to check for sequence similarities in their protein products.

*This article is located in the Journal of Cell Biology, Vol. 141, 1998



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