Blood, Vol. 93 No. 12 (June 15), 1999:
pp. 4300-4308
Factor XII Tenri, a Novel Cross-Reacting Material Negative Factor XII
Deficiency, Occurs Through a Proteasome-Mediated Degradation
Shinichi Kondo,
Fuminori Tokunaga,
Seiji Kawano,
Yoichi Oono,
Shunichi Kumagai, and
Takehiko Koide
From the Department of Life Science, Faculty of Science, Himeji
Institute of Technology, Harima Science Garden City, Hyogo; the
Department of Clinical Medicine, Kobe University, Kobe; and the
Department of Hematology, Tenri Yorozu Hospital, Tenri, Japan.
A homozygous cross-reacting material negative factor XII-deficient
patient with 3% antigen and activity levels of factor XII was screened
for the identification of a mutation at the genomic level. Low-ionic
strength single-stranded conformation polymorphism (SSCP) analysis and
sequence analysis showed that the proband's gene for factor XII had an
A
G substitution at nucleotide position 7832 in exon 3, resulting in a Tyr34 to Cys substitution in the NH2-terminal type II domain of factor XII. We designated
this mutation as factor XII Tenri. Mutagenic polymerase chain reaction (PCR), followed by KpnI digestion, showed a homozygous mutation in the proband's gene and heterozygous mutations in his parents and
sister. Immunoprecipitation and Western blot analyses of plasma samples
from the factor XII Tenri family indicated that the proband had a trace
amount of variant factor XII with an apparent molecular mass of 115 kD,
which was converted to the normal 80-kD form after reduction,
suggesting that factor XII Tenri was secreted as a disulfide-linked
heterodimer with a 
35-kD protein, which we identified as
1-microglobulin by immunoblotting. Pulse-chase
experiments using baby hamster kidney (BHK) cells showed that
Tenri-type factor XII was extensively degraded intracellularly, but the
addition of cystine resulted in increased secretion of the mutant.
Using membrane-permeable inhibitors, we observed that the degradation occurred in the pre-Golgi, nonlysosomal compartment and a proteasome appeared to play a major role in this process. On the basis of these in
vitro results, we speculate that the majority of the factor XII Tenri
is degraded intracellularly through a quality control mechanism in the
endoplasmic reticulum (ER), and a small amount of factor XII Tenri that
formed a disulfide-linked heterodimer with
1-microglobulin is secreted into the blood stream.
