Molecular cloning of a human apoC-Ill variant : Thr 74-+ Ala 74 mutation prevents 0-glycosylation

Apolipoprotein C-I11 (apoC-111) is a major protein of very low density lipoprotein (VLDL). The apoC-I11 polypeptide contains a carbohydrate chain containing galactosamine, galactose, and sialic acid attached in 0-linkage to a threonine residue at position 74. We have cloned the apoC-I11 gene from a subject whose serum contained unusually high amounts of apoC-111 lacking the carbohydrate moiety (C-111-0). DNA sequence analysis of the cloned gene revealed a single nucleotide substitution (A + G) that encodes an alanine at position 74 instead of the normal threonine. As a result ofthis amino acid replacement, the mutant apoC-111 polypeptide is not glycosylated. The mutation in the apoC-I11 gene creates a novel AluI site that permits diagnosis of the change by Southern blotting of genomic DNA. -Maeda, H. , R. K. Hashimoto, T. Ogura, S. Hiraga, and H. Uzawa. Molecular cloning of a human apoC-I11 variant: Thr 74 * Ala 74 mutation prevents 0glycosylation. J. Lipid Rcs. 1987. 28: 1405-1409. Supplementary key w o r d s gene gene doning apoC-111 mutant apolipoprotein apoC-111 gene human Apolipoprotein C-I11 (apoC-111) is a major protein constituent of the triglyceride-rich lipoproteins, chylomicrons, and very low density lipoproteins (VLDL), and a minor constituent of high density lipoprotein (HDL) in human serum. The precise physiological function of apoC-I11 in lipoprotein metabolism is not known. However, apoC-I11 has been shown to inhibit the in vitro activity of lipoprotein lipase (1) and to inhibit the apoEmediated uptake of triglyceride-rich lipoproteins by liver cells (2, 3). The preprotein of apoC-I11 is primarily synthesized in liver cells (4-6), and consists of 99 amino acids including a 20 amino acid signal sequence. After removal of the signal sequence, the mature apoC-I11 is modified by the attachment of a carbohydrate chain consisting of galactosamine, galactose, and sialic acid. The molar ratio of carbohydrate to apoC-I11 is 1 for galactosamine, 1 for galactose, and 0, 1, or 2 for sialic acid. The variations in the molar ratio of sialic acid to apoC-I11 result in three apoC-I11 polymorphic forms designated C-111-0, C-111-1, and C-111-2, respectively (7). We have previously described a family in which certain members contain unusually high amounts of unsialylated apoC-I11 (apoC-111-0) associated with their VLDL and HDL fractions (8). Family members having increased apoC-111-0 levels were asymptomatic and we demonstrated an autosomal-dominant Mendelian inheritance for this trait. Three possible mechanisms to explain the increase in unsialylated apoCI11 were postulated: first, reduction of the level of sialyltransferase; second, increase of a sialidase activity; and third, reduction in the activity andlor the number of asialoglycoprotein receptors (8). In the present study, we have cloned and sequenced the genomic DNA segments of the apoC-I11 gene from a member of the above family who had an increased level of serum apoC-111-0. We demonstrate that a single base substitution has occurred in the normal codon for threonine at position 74 that results in the replacement of threonine by alanine. The loss of the 0-linked sugar attachment site (Thr 74) thus explains the presence of an increased amount of apoC-111-0 in this subject. MATERIALS AND METHODS Electrophoresis of apoVLDL Venous blood was obtained after overnight fasting. Plasma VLDL particles (d < 1.006 glml) were isolated in a Type 50 rotor at 15OC, using a Beckman L5-50B ultracentrifuge. Polyacrylamide gel electrophoresis was carried out using 7.2% gels in 8 M urea. The gels were Abbreviations: VLDL, very low density lipoproteins; HDL, high density lipoproteins. ‘Present address and reprint requests to: Naruto University of Education, Faculty of Health and Living Sciences, Takashima-cho, Naruto City, Tokushima 772, Japan. Journal of Lipid Research Volume 28, 1987 1405 by gest, on N ovem er 6, 2017 w w w . j.org D ow nladed fom fixed and stained overnight with 0.003% Coomassie Brilliant Blue R in 10% trichloroacetic acid. same fragments were subcloned into the bacteriophage Ml3mplO and Ml3mpll for vectors (11).