Molecular mechanism of type I congenital heparin cofactor (HC) II deficiency caused by a missense mutation at reactive P2 site: HC II Tokushima.
We found a 66-year-old Japanese patient with type I congenital heparin cofactor (HC) II deficiency manifesting multiple atherosclerotic lesions. To investigate the molecular pathogenesis of our patient, we performed sequencing analysis and expressed recombinant human wild-type and mutant HC II molecules in COS-1 and CHO-K1 cells. Sequencing analysis following amplification of each of all 5 exons and its flanking region showed a single C to T transition at nucleotide position 12,854 in exon 5, which changed a Pro443 codon (CCG) to Leu codon (CTG). Because this mutation generates a new Bhv I site, the Bbv I digestion pattern of the PCR-amplified exon 5 fragments from each family member was analyzed. In all cases, the patterns were consistent with the activities and antigen levels of plasma HC I1 in those members. Transient transfection, metabolic labeling and pulse-chase experiments followed by immunoprecipitation analysis showed that the recombinant mutant HC II molecules were secreted from COS-1 cells in reduced amounts compared with the wild-type, and that an enhanced intracellular association of the mutant molecules with a chaperone, GRP78/BiP, was observed in CHO-K1 cells. Northern blot analysis indicated that the mutant HC I1 mRNA was transcribed at a similar level as that of wild-type. Immunohistochemical staining of the transfected cells revealed that COS-1 cells expressing the mutant HC II molecules were stained mainly in the perinuclear area. We conclude that the impaired secretion of the mutant HC II molecules, due to intracellular degradation, is the molecular pathogenesis of type I congenital HC II deficiency caused by a Pro443 to Leu mutation at reactive P2 site.
Kanagawa
Y
Y
First Department of Internal Medicine, University of Tokushima School of Medicine, Japan.
Shigekiyo
T
T
Aihara
K
K
Akaike
M
M
Azuma
H
H
Matsumoto
T
T
D000368:Aged; D000818:Animals; D001161:Arteriosclerosis / Q000209:etiology / Q000235:genetics; D001665:Binding Sites / Q000235:genetics*; D019556:COS Cells; D002352:Carrier Proteins / Q000378:metabolism; D004252:DNA Mutational Analysis; D000091342:Endoplasmic Reticulum Chaperone BiP; D005192:Family Health; D005260:Female; D006360:Heat-Shock Proteins; D015844:Heparin Cofactor II / Q000172:deficiency* / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008297:Male; D018832:Molecular Chaperones / Q000378:metabolism; D020125:Mutation, Missense; D010375:Pedigree; D011516:Prothrombin / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D015842:Serine Proteinase Inhibitors / Q000172:deficiency / Q000235:genetics / Q000378:metabolism; D014162:Transfection
D002352:Carrier Proteins; D000091342:Endoplasmic Reticulum Chaperone BiP; C000717247:HSPA5 protein, human; D006360:Heat-Shock Proteins; D018832:Molecular Chaperones; D011994:Recombinant Proteins; D015842:Serine Proteinase Inhibitors; C051306:prothrombin Tokushima; D015844:Heparin Cofactor II; D011516:Prothrombin