Human cardiac troponin I: precise identification of antigenic epitopes and prediction of secondary structure

Ferrières, Gaëlle; Calzolari, Charles; Mani, Jean‐Claude; Laune, Daniel; Trinquier, S; Laprade, Michel; Larue, Catherine; Pau, Bernard; Granier, Claude

doi:10.1093/clinchem/44.3.487

Cited by 52 publications

(18 citation statements)

References 17 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…1), were synthesized on a cellulose membrane (Abimed, Langenfield, Germany) by the Spot technique 16. The synthesis was performed by using an ASP 412 spotter (Abimed), as previously described 17. The set of membrane‐bound peptides was probed by incubation with anti‐gastric mucin MAbs (diluted 1:100).…”

Section: Methodsmentioning

confidence: 99%

Mapping of two new epitopes on the apomucin encoded by MUC5AC gene: Expression in normal GI tract and colon tumors

Nollet¹,

Forgue‐Lafitte²,

Kirkham³

et al. 2002

Intl Journal of Cancer

View full text Add to dashboard Cite

Gastric M1 mucin has been defined by several monoclonal antibodies (MAbs) as an early marker of human 1,2 and rat 3 colonic carcinogenesis. The gastric M1 mucin and MUC5AC gene have been detected in the human precancerous colonic 4 -7 and pancreatic 8 -10 epithelium using immunohistology and in situ hybridization, respectively. In pancreatic fluids removed using fine-needle aspiration under computerized scan control, an immunoradiometric assay (IRMA) of M1 mucin 11 improves the diagnosis of mucinous cystadenomas, a precancerous lesion for which surgery was suggested. 10 On the other hand, the early expression of this M1 mucin during colonic carcinogenesis could give excellent tools (i) for a better understanding of the early events associated with precancerous stages, (ii) for detecting patients with high a risk of developing colonic and pancreatic cancers and (iii) for studying the protective effect of drugs on the progression of colonic carcinogenesis. 12 Consequently, a better understanding of the biochemical nature of the M1 epitopes will be of invaluable help in this process.Eight anti-gastric M1 mucin MAbs were selected using immunohistology on gastrointestinal mucosae fixed with ethanol. These MAbs immunoreact against mucus cells of normal surface gastric mucosae, colonic adenomas and macroscopically normal colonic mucosae adjacent to adenocarcinomas but not against normal colon. 4,5 Among the 8 MAbs against gastric M1 mucin described up to now, 2 of them 19M1 and 21M1, which recognized the same or neighboring epitopes, 5 immunoreacted against a recombinant protein encoded by the 3Ј region of the MUC5AC gene 13 when expressed in a baculovirus/insect cell system. During the selection of hybridoma cells secreting anti-gastric mucin MAbs, we have also isolated 3 other MAbs (463M, 589M and 62M) 5,14 staining mucins from mucus cells of surface gastric epithelium and goblet cells of colonic adenomas but with patterns different from those observed using the other anti-M1 MAbs. Consequently, these 3 MAbs were not classified as "anti-M1 antibodies." In our study, using transfection of small fragments of the 3Ј region of the MUC5AC cDNA into COS-7 cells, we have mapped 2 new mucin epitopes within the already known M1-f epitope (MAbs 19/ 21M1) 5 on the C-terminal region encoded by the MUC5AC gene. We determined the smallest cDNA fragment that encoded a peptide corresponding to each epitope. We then compared the immunohistologic pattern in normal gastrointestinal tract and colonic tumors (hyperplastic polyps, adenomas and mucosae adjacent to adenocarcinomas) of the 2 new mucin epitopes with that of the M1-f epitope. MATERIAL AND METHODS Mucin antigensHuman gastric M1 mucin preparation. Mucin was isolated from mucinous ovarian cyst liquids or by scraping human gastric mucosa and purified using CsCl gradient and Sepharose chromatography, as already described. 11 Recombinant MUC5AC mucins. Two fusion proteins containing the secretory alkaline phosphatase (SeAP) at its N-terminal end and MUC5AC cDNAs 3Ј-coding sequence a...

show abstract

Section: Methodsmentioning

confidence: 99%

Mapping of two new epitopes on the apomucin encoded by MUC5AC gene: Expression in normal GI tract and colon tumors

Nollet¹,

Forgue‐Lafitte²,

Kirkham³

et al. 2002

Intl Journal of Cancer

View full text Add to dashboard Cite

show abstract

“…Ninety‐six overlapping 20‐mer peptides frameshifted by two residues, representing the complete hcTnI protein sequence [13], were synthesized on a cellulose membrane (Abimed GmbH, Langenfield, Germany) by the Spot technique [10]. The synthesis was performed by using an ASP 412 spotter (Abimed GmbH), as previously described [14].…”

Section: Methodsmentioning

confidence: 99%

“…Peptides were prepared by Fmoc solid‐phase synthesis on a AMS 422 robot (Abimed Gmbh) [15] as described [14]. Phosphorylated serine (S (p) ) was from Novabiochem as well as other Fmoc amino acids.…”

Section: Methodsmentioning

confidence: 99%

Systematic mapping of regions of human cardiac troponin I involved in binding to cardiac troponin C: N‐ and C‐terminal low affinity contributing regions

et al. 2000

Self Cite

View full text Add to dashboard Cite

The Spot method of multiple peptide synthesis was used to map in a systematic manner regions of the human cardiac troponin I sequence (hcTnI) involved in interactions with its physiological partner, troponin C (cTnC). Ninety-six 20-mer peptides describing the entire hcTnI sequence were chemically assembled; their reactivity with [125 I]cTnC, in the presence of 3 mM Ca 2+ , enabled the assignment of six sites of interaction (residues 19^32, 45^54, 129^138, 145^164, 161^178 and 1912 10). For several sites, a good correlation with literature data was obtained, thus validating this methodological approach. Synthetic peptides, each containing in their sequence an interaction site, were prepared. As assessed by BIACORE, all of them exhibited an affinity for cTnC in the range of 10 361 0 37 M, except for hcTnI [39^58] which showed a nanomolar affinity. This peptide was also able to block the interaction between hcTnI and cTnC. We therefore postulate that despite the existence of multiple cTnC interaction sites on the hcTnI molecule, only that region of hcTnI allows a stabilization of the complex. Residues 19^32 from the N-terminal cardio-specific extension of hcTnI were also found to be involved in interaction with cTnC; residues 19^32 may correspond to the minimal sequence of the extension which could switch between the N-and C-terminal TnC domains, depending on its phosphorylation state. Finally, two Ca 2+ -dependent cTnC binding domains within the C-terminal part of hcTnI (residues 164^178 and 191^210) were also mapped. The latter site may be linked with the cardiac dysfunction observed in stunned myocardium. ß

show abstract

“…Ferrieres et al . mapped several immunoreactive epitopes of the human‐cardiac TnI isoform, and also predicted secondary structure (and by exclusion, unstructured regions) 37. They found several epitopes that bound to cardiac‐specific N‐terminal extension (cNp), which they also predicted to be unstructured.…”

Section: Introductionmentioning

confidence: 99%

Isoform‐specific variation in the intrinsic disorder of troponin I

Hoffman

Sykes

2008

Proteins

View full text Add to dashboard Cite

Various intrinsic disorder (ID) prediction algorithms were applied to the three tissue isoforms of troponin I (TnI). The results were interpreted in terms of the known structure and dynamics of troponin. In line with previous results, all isoforms of TnI were predicted to have large stretches of ID. The predictions show that the C-termini of all isoforms are extensively disordered as is the N-terminal extension of the cardiac isoform. Cardiac TnI likely belongs to the group of intrinsically disordered signalling hub proteins. For a given portion of the protein sequence, most ID prediction approaches indicate isoform-dependent variations in the probability of disorder. Comparison of machine learning and physically based approaches suggests the ID variations are only partially attributable to local variations in the ratio of charged to hydrophobic residues. The VSL2B algorithm predicts the largest variations in ID across the isoforms, with the cardiac isoform having the highest probability of structured regions, and the fast-skeletal isoform having no intrinsic structure. The region corresponding to residues 57-95 of the fast-skeletal isoform, known to form a coiled coil substructure with troponin T, was highly variable between isoforms. The isoform-specific ID variations may have mechanistic significance, modulating the extent to which conformational fluctuations in tropomyosin are communicated to the troponin complex. We discuss structural mechanisms for this communication. Overall, the results motivate the development of predictors designed to address relative levels of disorder between highly similar proteins.

show abstract

Human cardiac troponin I: precise identification of antigenic epitopes and prediction of secondary structure

Cited by 52 publications

References 17 publications

Mapping of two new epitopes on the apomucin encoded by MUC5AC gene: Expression in normal GI tract and colon tumors

Mapping of two new epitopes on the apomucin encoded by MUC5AC gene: Expression in normal GI tract and colon tumors

Systematic mapping of regions of human cardiac troponin I involved in binding to cardiac troponin C: N‐ and C‐terminal low affinity contributing regions

Isoform‐specific variation in the intrinsic disorder of troponin I

Contact Info

Product

Resources

About