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Cloning, characterization, and analysis of the complete bovine factor XIIa inhibitor cDNA Edmeston, John Thorne
Abstract
The bovine homologue of the human Cl esterase inhibitor is called the bovine factor Xlla inhibitor. Like the human Cl esterase inhibitor, bovine factor Xlla inhibitor is a serine protease inhibitor with a reported molecular weight of between 72,000 and 92,000 Da. To determine the primary structure of bovine factor Xlla inhibitor, the cDNA was cloned and sequenced. Bovine total liver RNA was reverse transcribed using an oiigodTn primer to produce cDNA. This single stranded cDNA was then used as a template for polymerase chain reaction amplification experiments. The primers were designed using amino acid sequence data from tryptic fragments of purified plasmaderived bovine factor Xlla inhibitor and human Cl esterase inhibitor. The bovine factor Xlla inhibitor cDNA was amplified in two overlapping pieces and each piece was cloned separately. The cDNA sequence is 1608 bp long. The identity of the bovine factor Xlla inhibitor cDNA was established by comparison of the cDNA sequence with sequences derived from the bovine and human protein and the human cDNA. The 5-prime end of the cDNA starts at the initiation codon, ATG. The 3-prime end extends 179 base pairs past the stop codon, TAG, to the polyadenylation recognition signal AAT AAA, followed 14 bp later by the poly-A tail. An open reading frame of 1404 bp encodes the bovine factor Xlla inhibitor polypeptide. Translation of the bovine factor Xlla inhibitor cDNA nucleotide sequence into amino acid sequence results in a 468 amino acid polypeptide that includes a 23 amino acid leader peptide. The theoretical molecular weight of the 445 amino acid non-glycosylated protein, is 49,217 Da. Comparison of the predicted amino acid sequences of the bovine factor Xlla inhibitor and human Cl esterase inhibitor polypeptides shows an overall sequence identity of 60%. Both the human and bovine proteins have leader peptides, 22 amino acids and 23 amino acids respectively, which are 90% identical. The N-terminal region of the plasma bovine factor Xlla inhibitor, comprising the next 99 amino acids, shows only 17% identity with the same region of the human CI esterase inhibitor. The remainder of the proteins encode the serpin regions and share 68% identity. The mature human protein is 33 amino acids longer than the bovine protein; these extra residues occur as two insertions or amplifications with respect to the bovine protein. The first lies between residues 95 and 96 of the bovine protein and is 27 amino acids long; the second lies between residues 385 and 386 of the bovine protein and is 6 amino acids long. The 27 amino acid insertion comprises the bulk of a region in the human protein consisting of nine tetra-peptide repeats, seven of which conform to the consensus Glx-Pro-Thr-Thr. This region, in the human C1INH protein is associated with O-linked glycosylation. The six amino acid insertion lies at the very 3-prime end of exon 7 of the human gene. There is not enough data at this time to interpret the significance of the six amino acid insertion. It was unclear from the cDNA sequence if the 99 amino acid N-terminal region of the bovine factor Xlla inhibitor protein was merely a region of low identity or a novel exon that had been acquired by the bovine protein or deleted from the human protein. Experiments were performed to elucidate the genomic organization of the 5' region of the bovine factor Xlla inhibitor gene. The results show that while the N-terminal sequences of the human and bovine proteins are non-identical, the genomic organization in the 5- prime region of both genes is the same. Thus, the 27 amino acid insertion is the result of an amplification or deletion of an existing sequence rather than the acquisition or loss of an (novel) exon.
Item Metadata
Title |
Cloning, characterization, and analysis of the complete bovine factor XIIa inhibitor cDNA
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Creator | |
Publisher |
University of British Columbia
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Date Issued |
1995
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Description |
The bovine homologue of the human Cl esterase inhibitor is called the bovine
factor Xlla inhibitor. Like the human Cl esterase inhibitor, bovine factor Xlla inhibitor is
a serine protease inhibitor with a reported molecular weight of between 72,000 and
92,000 Da. To determine the primary structure of bovine factor Xlla inhibitor, the cDNA
was cloned and sequenced. Bovine total liver RNA was reverse transcribed using an
oiigodTn primer to produce cDNA. This single stranded cDNA was then used as a
template for polymerase chain reaction amplification experiments. The primers were
designed using amino acid sequence data from tryptic fragments of purified plasmaderived
bovine factor Xlla inhibitor and human Cl esterase inhibitor. The bovine factor
Xlla inhibitor cDNA was amplified in two overlapping pieces and each piece was cloned
separately. The cDNA sequence is 1608 bp long. The identity of the bovine factor Xlla
inhibitor cDNA was established by comparison of the cDNA sequence with sequences
derived from the bovine and human protein and the human cDNA. The 5-prime end of the
cDNA starts at the initiation codon, ATG. The 3-prime end extends 179 base pairs past
the stop codon, TAG, to the polyadenylation recognition signal AAT AAA, followed 14 bp
later by the poly-A tail. An open reading frame of 1404 bp encodes the bovine factor Xlla
inhibitor polypeptide. Translation of the bovine factor Xlla inhibitor cDNA nucleotide
sequence into amino acid sequence results in a 468 amino acid polypeptide that includes a
23 amino acid leader peptide. The theoretical molecular weight of the 445 amino acid
non-glycosylated protein, is 49,217 Da. Comparison of the predicted amino acid
sequences of the bovine factor Xlla inhibitor and human Cl esterase inhibitor
polypeptides shows an overall sequence identity of 60%. Both the human and bovine
proteins have leader peptides, 22 amino acids and 23 amino acids respectively, which are
90% identical. The N-terminal region of the plasma bovine factor Xlla inhibitor,
comprising the next 99 amino acids, shows only 17% identity with the same region of the
human CI esterase inhibitor. The remainder of the proteins encode the serpin regions and
share 68% identity. The mature human protein is 33 amino acids longer than the bovine
protein; these extra residues occur as two insertions or amplifications with respect to the
bovine protein. The first lies between residues 95 and 96 of the bovine protein and is 27
amino acids long; the second lies between residues 385 and 386 of the bovine protein and
is 6 amino acids long. The 27 amino acid insertion comprises the bulk of a region in the
human protein consisting of nine tetra-peptide repeats, seven of which conform to the
consensus Glx-Pro-Thr-Thr. This region, in the human C1INH protein is associated with
O-linked glycosylation. The six amino acid insertion lies at the very 3-prime end of exon 7
of the human gene. There is not enough data at this time to interpret the significance of
the six amino acid insertion.
It was unclear from the cDNA sequence if the 99 amino acid N-terminal region of
the bovine factor Xlla inhibitor protein was merely a region of low identity or a novel
exon that had been acquired by the bovine protein or deleted from the human protein.
Experiments were performed to elucidate the genomic organization of the 5' region of the
bovine factor Xlla inhibitor gene. The results show that while the N-terminal sequences
of the human and bovine proteins are non-identical, the genomic organization in the 5-
prime region of both genes is the same. Thus, the 27 amino acid insertion is the result of
an amplification or deletion of an existing sequence rather than the acquisition or loss of
an (novel) exon.
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Extent |
6033492 bytes
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Genre | |
Type | |
File Format |
application/pdf
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Language |
eng
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Date Available |
2009-01-19
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Provider |
Vancouver : University of British Columbia Library
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Rights |
For non-commercial purposes only, such as research, private study and education. Additional conditions apply, see Terms of Use https://open.library.ubc.ca/terms_of_use.
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DOI |
10.14288/1.0086826
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URI | |
Degree | |
Program | |
Affiliation | |
Degree Grantor |
University of British Columbia
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Graduation Date |
1995-11
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Campus | |
Scholarly Level |
Graduate
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Aggregated Source Repository |
DSpace
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Item Media
Item Citations and Data
Rights
For non-commercial purposes only, such as research, private study and education. Additional conditions apply, see Terms of Use https://open.library.ubc.ca/terms_of_use.