Epitope of titin A-band-specific monoclonal antibody Tit1 5 H1.1 is highly conserved in several Fn3 domains of the titin molecule. Centriole staining in human, mouse and zebrafish cells
© Mikelsaar et al.; licensee BioMed Central Ltd. 2012
Received: 14 March 2012
Accepted: 14 September 2012
Published: 17 September 2012
Previously we have reported on the development of a new mouse anti-titin monoclonal antibody, named MAb Titl 5 H1.1, using the synthetic peptide N-AVNKYGIGEPLESDSVVAK-C which corresponds to an amino acid sequence in the A-region of the titin molecule as immunogen. In the human skeletal muscles, MAb Titl 5 H1.1 reacts specifically with titin in the A-band of the sarcomere and in different non-muscle cell types with nucleus and cytoplasm, including centrioles. In this report we have studied the evolutionary aspects of the binding of MAb Tit1 5 H1.1 with its target antigen (titin).
We have specified the epitope area of MAb Tit1 5 H1.1 by subpeptide mapping to the hexapeptide N-AVNKYG-C. According to protein databases this amino acid sequence is located in the COOH-terminus of several different Fn3 domains of the A-region of titin molecule in many organisms, such as human being, mouse, rabbit, zebrafish (Danio rerio), and even in sea squirt (Ciona intestinalis). Our immunohisto- and cytochemical studies with MAb Tit1 5 H1.1 in human, mouse and zebrafish tissues and cell cultures showed a striated staining pattern in muscle cells and also staining of centrioles, cytoplasm and nuclei in non-muscle cells.
The data confirm that titin can play, in addition to the known roles in striated muscle cells also an important role in non-muscle cells as a centriole associated protein. This phenomenon is highly conserved in the evolution and is related to Fn3 domains of the titin molecule. Using titin A-band-specific monoclonal antibody MAb Tit1 5 H1.1 it was possible to locate titin in the sarcomeres of skeletal muscle cells and in the centrioles, cytoplasm and nuclei of non-muscle cells in phylogenetically so distant organisms as Homo sapiens, Mus musculus and zebrafish (Danio rerio).
We have reported previously  on the development of a new mouse anti-titin monoclonal antibody, named MAb Titl 5 H1.1, using the synthetic peptide N-AVNKYGIGEPLESDSVVAK-C corresponding to an amino acid sequence in the A-band of the titin molecule as immunogen. In the human skeletal muscle, MAb Tit1 5 H1.1 reveals a clearly striated staining pattem, reacting with the A-band of the sarcomere. The antibody reacts with titin in cytoplasm, nucleus and centrioles in all of non-muscle cell types investigated so far. In the present study we have restricted (narrowed down) the epitope of MAb Tit1 5 H1.1 to the hexapeptide N-AVNKYG-C by subpeptide mapping and performed immunohisto-and cytochemically studies of the of MAb Tit1 5 H1.1 epitope expression in phenotypically distant organisms such are human being Homo sapiens, mouse Mus musculus and zebrafish (Danio rerio).
Results and discussion
Specifying the epitope of MAb Tit1 5 H1.1 by subpeptide mapping
Protein database analysis of the evolutionary relationship of the „narrowed”epitope AVNKYG of MAb Tit1 5 H1.1
To investigate the evolutionary relationship of the narrowed amino acid epitope (AVNKYG) of MAb Tit1 5 H1.1 between different organisms, the analysis of the information stored in protein databases was performed. The amino acid sequence N-AVNKYG-C was present in the C-terminus of several Fn3 domains of the human (Q8WZ42), mouse (A2ASS6), rabbit (Q28733-titin fragment), zebrafish (Danio rerio) (titin a) and even in sea squirt (Ciona intestinalis-UPI000180D3B0) titin molecule. (A blast sequence analysis of human proteins revealed the hexapeptide N-AVNKYG-C with 100% query coverage and 100% maximal identity only in 15 human titin isoforms, and 2 protein fragments of unknown origin which seem to be also fragments of titin molecule). We have used SMART:Fn3 domain annotation database for the numeration of Fn3 domains because according to that the first Fn3 domains from the N-terminus side of the titin molecule are fully comparable between the three species studied. In Protein Knowledgebase UniProtKB the number of Fn3 domains in the mouse (A2ASS6) is 134, and the numeration starts with No.1 in position 942–1037 and No. 2 in position 14343–14433. However, in SMART:FN3 domain annotation database these Fn3 domains do not exist at all, and our analysis has also shown that these Fn3 domains No. 1 and No.2 in the mouse do not have any amino acid homology either with human or zebrafish first Fn3 domains. According to SMART:FN3 domain annotation database the numeration of Fn3 domains can start from the No.1 in all of the three species, and the amino acid sequences of the corresponding Fn3 domains are fully comparable. However, in the zebrafish (Danio rerio) the Fn3 domain corresponding to Fn3 domain No.26 is fully absent, and so the numbers of the following Fn3 domains of the zebrafish are by one number lower compared to human and mouse titin. So, human and mouse Fn3 domain No.27 should be compared with the Fn3 domain number 26 in the zebrafish, and so on.
Comparison of amino acid sequences between the corresponding Fn3 domains containing AVNKYG sequence in human (Q8WZ42),mouse (A2ASS6) and zebrafish (A5X6X5) titin molecule
No’s of Fn3, species and location of Fn3 domains
Amino acid sequences of Fn3 domains of titin. The data according to SMART: Fn3 annotation http://smart.embl.de/smart/do_annotation.pl?DOMAIN=SM00060
PS PPRN L AVTD IKAESC Y L T WDAPLD NGGSE I T H YVIDKR D ASRKKA EWE E VTNTAV E K RYG I W K L IPN G QYE FRV R AVNKYG IS
PS PPRN L AVTD IKAESC Y L TWDAPLD N GGSE I T H YIIDKR D ASRKKS EWEE VTNTAV E R RYG I W K L IPN G QYE FRV R AVNKYG IS
PT PPRN V AVSS IKAESCN L S WDAPLD I GGSEL T N YIVEMKD LNVEDP E KA E WVQVTKSII EK RYGVWN L VTG G NYKFRV K A ENKYG IS
PGPPEGPL A V TEV T S EKCVLSW F PPL D DGG A KI DH YI V Q K RETSRL A WTNVA SEV QV TKL KVTKLLKGNEYIFRV K AVNKYG VG
PGPPEGPL A V SDV T S EKCVLSW L PPL D DGG A KI DH YI V Q K RETSRL A WTNVA TEV QV TKL KVTKLLKGNEYIFRV M AVNKYG VG
PGPPEGPL H V TDM T V EKCVLSW L PPL H DGG G KI EY YI I Q R RETSRL T WTNVA TDL QV NRY KVTKLLKGNEYIFRV M AVNKYG VG
PGPP E GP VV I S GVT A EKC TLA WK P PL Q DGG SDIIN YIVERRETSRLVWT V V DAN VQTL SCKVT KLL E GNEY T FR IM AVNKYG V G
PGPP E GP VA I S GVT AEKC TLA WK P PL Q DGG SDITN YIVERRETSRLVWT L V DAN VQTL SCKVL KLL E GNEY I FR IM AVNKYG V G
PGPP D GP IS I Y GVT S EKC CIS WK T PL H DGG AEVSH YIVERRETSRLVWT V V ELK VQTL NLK IT KLL P GNEY I FR VI PVNKYG I G
PGPPEGP VQ V T GVT S EKC S L T W S PP LQ DGGS D IS H YV VE KRETSRLAWTVV ASEVVTNSL KVTKLL E GN E YV FR I M AVNKYG VG
PGPPEGP VQ V T GVT A EKC T L A W S PP LQ DGGS DIS H YV VE KRETSRLAWTVV ASEVVTNSL KVTKLL E GN K Y I FR I M AVNKYG VG
PGPPEGP LT V S GVT N EKC S L S W L PP RH DGGS S IS Y YV IQ KRETSRLAWTVV SGDCGATMFKVTKLL KGN E Y I FR VM AVNKYG VG
PGPP E GPLKVTGV T AEKCYL A W NP P LQ DGGA N ISHYIIEKRETSRLSWT Q V STEV QA LN YKVTKLLPGNEYIFRVM AVNKYG IG
PGPP E GPLKVTGV T AEKCYL A W NP P LQ DGGA S ISHYIIEKRETSRLSWT Q V SNEV QA LN YKVTKLLPGNEYIFRVM AVNKYG IG
PGPP D GPLKVTGV A AEKCYL H W SH P SH DGGA S ISHYIIEKRETSRLSWT V V EPKI QA IS YKVTKLLPGNEYIFRVM AVNKYG IG
PGPP GGP I E FK V TA EKI T LL W R PPAD D GGA KI THYIVEKRETSR VV WS MVS E HLEE CI IT TTKI IKGNEY I FRVR AVNKYG I G
PGPP GGP I E FK V TA EKI T L LW R PPAD D GGA KI THYIVEKRETSR VV WS MVA E NLEE CI T TTKI IKGNEY V FRVR AVNKYG I G
PGPP AGE I Q FK I TA DTM T IM W D PPAD E GGA MV THYIVEKRETSR IM WS IIS E KLQD CI T VPRLIKGNEY I FRVR GVNK HG VG
PGP CGKLTVS RVT Q EKCT LA W SL P Q EDGG AEIT HYIVERRETSRLNWVI V E G EC P T L S Y V VTR LIKNNEY I FRVR AVNKYG PG
PGP CGKLTV RVT E EKCT LA W S LP Q EDGG AEIT HYIVERRETSRLNWVI V E G EC L T A S Y V VTR LIKNNEY T FRVR AVNKYG LG
PGP PAGTITIS RVT D EKCT VS W K I P L EDGG DHVS HYIVERRETSRLNWVI M E T EC K T L S C V STK LIKNNEY I FRVR GVNKYG PG
Amino acid sequence homology (in%) between the corresponding Fn3 domains containing AVNKYG sequence in human (Q8WZ42),mouse (A2ASS6) and zebrafish (A5X6X5) titin molecule
The homology beween AVNKYG containing Fn3 domains in human (Q8WZ42), mouse (A2ASS6) and zebrafish ( Danio rerio - A5X6X5) titin molecule
Immunohistochemistry of human and zebrafish skeletal muscle biopsies using MAb Tit1 5 H1.1
Immunocytochemistry of cultured human, mouse and zebrafish (Danio rerio) cells
In conclusion, we have succeeded in narrowing down the peptide epitope of our titin A-band-specific monoclonal antibody Tit1 5 H1.1 from 19-aa-peptide N-AVNKYGIGEPLESDSVVAK-C to hexapeptide N-AVNKYG-C. This peptide sequence proved to be highly conserved in several corresponding Fn3 domains of titin in different organisms. It was possible by using MAb Tit1 5 H1.1 to locate titin immunohisto-and cytochemically both in the sarcomeres of skeletal muscle cells and in centrioles, cytoplasm and the nuclei of non-muscle cells in phylogenetically so distant organisms as are Homo sapiens, Mus musculus and zebrafish (Danio rerio). These findings prove that titin has been a very ancient component of the centrosome.
Materials and methods
Human cell cultures
In this study an original normal human adult skin cell culture SA-54 (developed by LabAs Ltd.) and the commercial cell-line human melanoma Bowes were used. The cell lines were grown in a medium containing DMEM/F12 supplemented with 10% of FCS and gentamycin (all Invitrogen, GIBCO).
Mouse cell cultures
Mouse embryonic fibroblast culture MEF7 was used and prepared according to the prescription of Boris Greber (Isolation and handling of primary mouse embryonic fibroblasts (MEFs) accompanying protocol to “Mouse embryonic stem (ES) cell culture - basic procedures” http://www.molgen.mpg.de/~rodent/MEF_protocol.pdf).
Zebrafish skeletal muscle biopsies, and short-term cell cultures of non-muscle cells
Zebrafish ( Danio rerio )
Adult Zebrafishes (Danio rerio) were purchased from a licensed zooshop.
The research was approved under animal care permit No.102 by the Commission of the Authorization of Animal Testing Permits of the Estonian Ministry of Agriculture.
Zebrafish organs for immuno-and cytochemistry
Fishes were fasted for at least 24 h, anesthetized in 0.2% Tricaine (ethyl 3-aminobenzoate methanesulfonate salt, Tricaine MS-222, Fluka, cat.no. A5040) and then euthanized by incubation in ice water for 15 min. Tricaine was added directly to the anaesthetic bath. An euthanized male fish was rinsed once with 70% ethanol and placed into a sterile PBS solution with antibioticum gentamycin in a Petri dish. The skin and muscle of the fish were carefully removed from the ventral wall and internal organs visualized as described by Gupta and Mullins . Spinal muscle tissue biopsies were taken for immunohistochemistry and testes were removed for a short-term cell culture.
Short-term cultures of zebrafish testes
The testes were removed and placed into a sterile Petri dish with sterile PBS supplemented with gentamycin. Thereafter testes were minced and trypsinized for 5 min with a mixture of 0.05% trypsin with 0.53 mM EDTA. The cell suspension (suspension contained also some small cell clusters) was washed for 3 times by centrifugation at 100 g for 5 min with DMEM:F12 medium containing 10% of FCS and gentamycin (Gibco, Invitrogen) and seeded to grow on the cover-glasses in 6-well plates (Nunc) in DMEM:F12 medium containing 20% of FCS and gentamycin (all Invitrogen, GIBCO). The cells were cultured in CO2 incubators at 37°C for 1–3 days.
Immunohistochemistry of zebrafish skeletal muscle
Zebrafish skeletal muscle cryoslices were prepared from skeletal muscle biopsies using the embedding medium (Thermo Shandon, Pittsburgh,PA). Sections were cut by using cryostat Cryocute E. Reichert-Jung at a thickness of between 8 and 10 μm. The sections were fixed with 4% PFA in PBS for 30 min at 4°C, washed for 3×5 min with PBS and incubated with the hybridoma supernatant of anti-titin MAb Titl 5 H1.1 for 1 h at RT, then washed for 3×5 min with PBS. The reaction was visualized by using Alexa 594 or A488 fluorochrome-conjugated goat secondary anti-mouse IgG antibody (Invitrogen, Molecular Probes, Eugene, Oregon, USA). The cell nuclei were counterstained with DAPI. The preparations were mounted in the anti-fading mounting medium Prolong Gold Antifade (Molecular Probes) and covered with coverslips. The immunoreaction was checked by a visual microscoping system (Olympus BX, using objectives UplanFI 20x/0.50, 40x/0.75, or 100x/1.30 Oil Iris, and the Olympus DP50-CU Photographing System, Tokyo, Japan).
Immunocytochemistry of cells grown in vitro
Paraformaldehyde (PFA) fixation method was used. In PFA fixation the coverslips with the growing cells were transferred without any previous washing into dishes containing pre-warmed 4% paraformaldehyde in PBS and left for 5 min at room temperature. Then the coverslips were washed three times for 5 min each with PBS, and the excess of aldehyde was quenched with 50 mM NH4Cl in PBS (10 min). After washing twice with PBS, the cells were permeabilized for 10 min with 0.1% Tritone X-100 in PBS, washed with PBS and blocked. The coverslips were then transferred into a blocking solution (0.3% casein, 0.01% Tween-20 in PBS) for 1 h at room temperature or overnight at 4°C. The blocking solution was removed by aspiration, and the cells were stained as follows. The cells were incubated for 1 h at RT with the MAb Tit1 5 H1.1 supernatant. The coverslips were washed at least three times for 5 min each with PBS, and immunolabeling was visualized by incubating the cells with the secondary goat anti-mouse antibody conjugated with fluorochrome Alexa 594 (cat.no. A11032, Molecular Probes) for 1 h at RT. In all cases, the coverslips were washed at least three times for 5 min with PBS, 10 μL of DAPI solution (1 mg/mL) was added into the last PBS, and then the coverslips were incubated for 5 min at RT. After quick rinsing in distilled water, the coverslips were mounted in the anti-fading mounting medium Prolong Gold Antifade (Molecular Probes). The cells were checked by a visual microscoping system (Olympus BX, using objectives UplanFI 20x/0.50, 40x/0.75, or 100x/1.30 Oil Iris, and Olympus DP50-CU Photographing System). Phalloidin conjugated to Alexa 488 (Molecular probes) was used as marker for F-actin (Molecular Probes). The following primary antibodies were used for the immunofluorescence co-localization of the target antigen (titin) of MAb Tit1 5 H1.1 with centrioles/centrosomes: goat polyclonal antibody to human ninein and rabbit polyclonal antibody to human γ-tubulin (both from Santa Cruz Biotechnology,Inc., cat.no. sc-50142 and sc-10732, respectively), and rabbit polyclonal antibody to human pericentrin (cat.no. ab4448, AbCam, Cambridge, UK). The specific staining of ninein was visualized with Alexa 488 conjugated to donkey anti-goat secondary antibody (cat.no. A11055, Molecular Probes) and the specific staining of γ-tubulin and pericentrin was visualized with Alexa 488 conjugated to goat anti-rabbit secondary antibody (cat.no. A11008, Molecular Probes). We have used for the co-localization study of titin and γ-tubulin the same rabbit anti-human γ-tubulin polyclonal antibody (sc-10732, H-183, Santa Cruz Biotechnlogy, Inc.) that is developed against a synthetic peptide corresponding to last 183 amino acids in C-terminal part of human gamma-tubulin (tubulin gamma-1 chain, human, P23258, UniProtKB/Swiss-Prot) and used by us for human cells. This amino acid sequence has 96,2% of homology with zebrafish gamma-tubulin (tubulin gamma-like,Danio rerio, Q7ZVM5, UniProtKB/TrEMBL), and the anti-human gamma-tubulin antibody works well also in zebrafish cells.
Epitope analysis of MAb Tit1 5 H1.1 by subpeptide mapping
The amino acid sequence originally used to prepare anti-titin monoclonal antibody MAb Tit1 5 H1.1 was a 19-amino-acid-long peptide with the sequence (NH2)AVNKYGIGEPLESDSVVAK(COOH), which is for the most part located in the C-terminus of fibronectin type-III domain 103 of the A-band of a full titin molecule (SwissProt: Q8WZ42). To narrow down the epitope of MAb Tit1 5 H1.1 the following subpeptides were used: AVNKYG, IGEPLE, EPLESD, PLESDSV and ESDSVV. The peptides were synthesized by Inbio Ltd. (Tallinn, Estonia). Inhibition of MAb Tit1 5 H1.1 by subpeptides was performed incubating 1,0 mL of the supernatant of MAb Titl 5 H1.1 overnight with 2 mg of each peptide. This was considered to give ~ 100 x overweight of the peptide compared to the amount of MAb calculated to be 20 μg per 1 mL as a maximum. The supernatants with inhibited MAb Titl 5 H1.1 were used in immunofluorescence studies.
Protein database analysis of the evolutionary relationship of the “narrowed”epitope N-AVNKYG- C of MAb Tit1 5 H1.1
The following protein databases were used to investigate the evolutionary relationship of the narrowed amino acid epitope (AVNKYG) of MAb Tit1 5 H1.1 with other organisms: PROWL of the Rockefeller university http://prowl.rockefeller.edu/prowl/proteininfo.html, Protein Knowledgebase UniProtKB http://www.uniprot.org/, SMART:FN3 domain annotation http://smart.embl.de/smart/do_annotation.pl?DOMAIN=SM00060 and Wellcome Trust Sanger Institute http://pfam.sanger.ac.uk/.
A.V.M. is professor in human biology and genetics at the Institute of General and Molecular Pathology of Medical faculty of the University of Tartu, his main scientific interests are connected with human genetics, medical genetics, cell biology and immunobiotechnology.
AS (Mag of Sci) is a researcher in human biology and genetics at the Institute of General and Molecular Pathology of Medical faculty of the University of Tartu. His main scientific interests are connected with proteomics and cell biology.
RM ( MD, PhD) is Ass. Professor in medical genetics at the Institute of General and Molecular Pathology of Medical faculty of the University of Tartu. Her main scientific interests are connected with cytogenetics, medical genetics and cell biology.
PT (Mag Sci) is a researcher at Estonian Life Science University, specialist in proteomics and using of ESI-MS/MS.
AK is a specialist in immunohistochemistry at the Institute of General and Molecular Pathology of Medical faculty of the University of Tartu
IM is a laboratory specialist in the field of ichthyology at LabAs Ltd
EJ ( PhD) is a senior researcher, at the Institute of General and Molecular Pathology of Medical faculty of the University of Tartu. His main scientific interest are connected with cell biology, immunobiotechnology
This work was partly supported by target financing SF0188096s08 of the Estonian Ministry of Science and Education and grant no. 8981 from Estonian Science Foundation. The authors thank Dr. Siiri Altraja for the critical review of the manuscript.
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