Cloning of a newly identified heart-specific troponin I isoform, which lacks the troponin T binding portion, using the yeast hybrid system

Abstract

Objective: To elucidate the molecular pathogenesis behind increased levels of laminin in cardiac muscle cells in cardiomyopathy by using a yeast hybrid screen. The present study reports the cloning of a newly identified heart-specific troponin I isoform, which is putatively linked to laminin. Future studies will explore the functional significance of this connection.

Methods: Yeast two-hybrid screen analysis was performed using MLF1-interacting protein (amino acids 1 to 318) as bait. The human heart complementary DNA library was screened by using the yeast-mating method for overnight culture.

Results: Two final positive clones from the heart library were isolated. These two clones encoded the same protein, a short isoform of human cardiac troponin I (TnI) that lacked TnI exons 5 and 6. The TnI isoform has a heart-specific expression pattern and it shares several sequence features with human cardiac TnI; however, it lacks the troponin T binding portion.

Conclusion: The heart-specific segment of the human cardiac TnI isoform shares several sequence features with human cardiac TnI, but it lacks the troponin T binding portion. These results suggest that the heart-specific TnI isoform may be involved in cardiac development and disease.

Keywords: Hybrid system; TNNI3; Tropomyosin; Troponin I.

Figure 1)

The complete nucleotide and deduced amino acid sequence of the troponin I (TnI) isoform isolated from a human heart complementary DNA (cDNA) library. The open reading frame of MLF1-interacting protein (amino acids 1 to 318) was used as bait in two-hybrid screening to isolate molecular partners (15–17). The fragment of the cDNA clone was isolated from a human cardiac cDNA library. It contains a 5′ (132 base pair) and a long 3′ (90 base pair) untranslated region excluding the poly(A) tail. The encoded protein is 119 residues. Numbering of amino acids begins with the putative start codon which starts the longest open reading frame. The DNA sequence of each of the exon/intron boundaries of the human TnI and TnI isoform is shown (arrow)

Figure 2)

Alignment of the amino acid sequences of human cardiac troponin I (TnI) and the TnI isoform. The amino acid sequences of human cardiac TnI (top), the TnI isoform (bottom) and the consensus (middle) are aligned (5). Dashes mark the absence of an amino acid in the TnI isoform compared with human cardiac TnI

Figure 3)

Analyses of cloned troponin I (TnI) and human cardiac TnI messenger RNA in human tissues. Total RNA was electrophoresed on a formaldehyde-agarose gel (15 mg total RNA per lane) and blotted onto a nitrocellulose membrane. The blot was hybridized with a ³²P-labelled TnI complementary DNA probe and autoradiographed (upper panel). The same membrane was then stripped, rehybridized with a ³²P-labelled complementary DNA probe of the TnI isoform and autoradiographed (middle panel). 18S ribosomal RNA on the same membrane was stained using ethidium bromide as a loading control (bottom panel)

Figure 4)

Schematic representation of human cardiac troponin I (TnI) (top) and the TnI isoform (bottom) identified in the present study from a complementary DNA library using a yeast hybrid screen. The TnI isoform lacks amino acids located at positions 41 to 131 in TnI (the portion containing the troponin T [TnT] binding site). This isoform contains the inhibitory region of TnI (amino acids 130 to 148), which binds actin, and also contains a full C-terminal actin binding portion (amino acids 173 to 181). TnC Troponin C