Abstract
Genetic analysis of a heterozygous protein C-deficient patient revealed a novel deletion of a single guanine residue (8857G) among four consecutive guanine nucleotides [380TrP(TGG)-381Gly(GGT)] in exon IX, which encodes the carboxyl-terminal region of protein C. This deletion results in a frameshift mutation and substitution of the last 39 amino acids (381Gly-419Pro) with 81 abnormal amino acid residues, and we have designated this elongated variant as Protein C-Nagoya. A mutagenic primer was designed which replaced the third guanine residue upstream from the deletion with cytosine, thereby creating a new Aval site in an otherwise normal allele. Analysis of the polymerase chain reaction products derived from this, mutagenic primer showed that the abnormal allele has been inherited in this family. To elucidate how this molecular abnormality leads to protein C deficiency, an expression plasmid containing this mutation was transfected into COS 7, BHK, and ψ-2 cells, and the secretory process of the expressed Protein C-Nagoya was analyzed. ELISA and immunoprecipitation analysis with [35S]methionine labeling indicated that the mutant protein C, which was larger in size than normal, was mostly retained within the cells, and only a small portion of it was secreted into the medium. These results suggest that most of Protein C-Nagoya undergoes degradation within the producing cells, and this frameshift mutation apparently leads to protein C deficiency by impairment of secretion of the elongated protein C into plasma.
| Original language | English |
|---|---|
| Pages (from-to) | 2439-2446 |
| Number of pages | 8 |
| Journal | Journal of Clinical Investigation |
| Volume | 90 |
| Issue number | 6 |
| Publication status | Published - Dec 1992 |
| Externally published | Yes |
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Keywords
- Expression study
- Family diagnosis
- Frameshift mutation
- In vitro mutagenesis
- Thrombophilia
ASJC Scopus subject areas
- Medicine(all)
Cite this
Impaired secretion of the elongated mutant of protein C (protein C-nagoya) : Molecular and cellular basis for hereditary protein C deficiency. / Yamamoto, Koji; Tanimoto, Mitsune; Emi, Nobuhiko; Matsushita, Tadashi; Takamatsu, Junki; Saito, Hidehiko.
In: Journal of Clinical Investigation, Vol. 90, No. 6, 12.1992, p. 2439-2446.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Impaired secretion of the elongated mutant of protein C (protein C-nagoya)
T2 - Molecular and cellular basis for hereditary protein C deficiency
AU - Yamamoto, Koji
AU - Tanimoto, Mitsune
AU - Emi, Nobuhiko
AU - Matsushita, Tadashi
AU - Takamatsu, Junki
AU - Saito, Hidehiko
PY - 1992/12
Y1 - 1992/12
N2 - Genetic analysis of a heterozygous protein C-deficient patient revealed a novel deletion of a single guanine residue (8857G) among four consecutive guanine nucleotides [380TrP(TGG)-381Gly(GGT)] in exon IX, which encodes the carboxyl-terminal region of protein C. This deletion results in a frameshift mutation and substitution of the last 39 amino acids (381Gly-419Pro) with 81 abnormal amino acid residues, and we have designated this elongated variant as Protein C-Nagoya. A mutagenic primer was designed which replaced the third guanine residue upstream from the deletion with cytosine, thereby creating a new Aval site in an otherwise normal allele. Analysis of the polymerase chain reaction products derived from this, mutagenic primer showed that the abnormal allele has been inherited in this family. To elucidate how this molecular abnormality leads to protein C deficiency, an expression plasmid containing this mutation was transfected into COS 7, BHK, and ψ-2 cells, and the secretory process of the expressed Protein C-Nagoya was analyzed. ELISA and immunoprecipitation analysis with [35S]methionine labeling indicated that the mutant protein C, which was larger in size than normal, was mostly retained within the cells, and only a small portion of it was secreted into the medium. These results suggest that most of Protein C-Nagoya undergoes degradation within the producing cells, and this frameshift mutation apparently leads to protein C deficiency by impairment of secretion of the elongated protein C into plasma.
AB - Genetic analysis of a heterozygous protein C-deficient patient revealed a novel deletion of a single guanine residue (8857G) among four consecutive guanine nucleotides [380TrP(TGG)-381Gly(GGT)] in exon IX, which encodes the carboxyl-terminal region of protein C. This deletion results in a frameshift mutation and substitution of the last 39 amino acids (381Gly-419Pro) with 81 abnormal amino acid residues, and we have designated this elongated variant as Protein C-Nagoya. A mutagenic primer was designed which replaced the third guanine residue upstream from the deletion with cytosine, thereby creating a new Aval site in an otherwise normal allele. Analysis of the polymerase chain reaction products derived from this, mutagenic primer showed that the abnormal allele has been inherited in this family. To elucidate how this molecular abnormality leads to protein C deficiency, an expression plasmid containing this mutation was transfected into COS 7, BHK, and ψ-2 cells, and the secretory process of the expressed Protein C-Nagoya was analyzed. ELISA and immunoprecipitation analysis with [35S]methionine labeling indicated that the mutant protein C, which was larger in size than normal, was mostly retained within the cells, and only a small portion of it was secreted into the medium. These results suggest that most of Protein C-Nagoya undergoes degradation within the producing cells, and this frameshift mutation apparently leads to protein C deficiency by impairment of secretion of the elongated protein C into plasma.
KW - Expression study
KW - Family diagnosis
KW - Frameshift mutation
KW - In vitro mutagenesis
KW - Thrombophilia
UR - http://www.scopus.com/inward/record.url?scp=0027052583&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=0027052583&partnerID=8YFLogxK
M3 - Article
VL - 90
SP - 2439
EP - 2446
JO - Journal of Clinical Investigation
JF - Journal of Clinical Investigation
SN - 0021-9738
IS - 6
ER -