Identification of a Major Heparin-Precipitable Protein in Human Serum and Its Relationship to Cystic Fibrosis

Abstract

SummaryHeparin binding to serum proteins and their subsequent precip itation is reportedly increased in cystic fibrosis (CF). We have confirmed this finding for CF patients over the age of 12 i11.34 + 2.42 mg/ml precipitated protein for normals ( n = 19) versus 17.46 5 4.60 mg/ml for CF patients ( n = 37), P < 0.001; 0.629 f 0.098 mg/ml precipitated heparin for normals versus 0.789 + 0.206 for CF patients, P < 0.01j. We have also shown that patients with a variety of pulmonary diseases unrelated to CF do not show this effect. When the amounts of protein and heparin precipitated are compared with the amount of IgG found in the whole serum sample, the correlation coefficients (protein r = 0.77; heparin, 0.74; n = 81) are significant at a level of P < 0.001. In addition, the report that young CF patients exhibit hypogammaglobulinemia prompted us to examine serum samples from CF patients and agematched controls under the age of 12. No differences were found.These findings prompted us to identify the proteins precipitated from CF and control sera by heparin at pH 5.57. The literature provides little information, inasmuch as most serum protein-heparin interactions have been studied at neutral or basic pHs.Because of its polyanionic character, we anticipated that heparin would interact most strongly with positively charged serum proteins, i.e., the gamma globulins. IgG, which represents the bulk of this class of proteins, is elevated in serum samples from many patients with cystic fibrosis (12,13,18,21 The results of this study show that IgG is indeed the major protein precipitated by heparin at pH 5.57 and suggest that precipitation of increased amounts of IgG is largely responsible for previous observations of heparin interactions with CF serum components.To investigate the molecuiar basis for this effect, sera from patients with CF and from age-matched controls were precipitated MATERIALS AND METHODS with 50 mg% heparin at pH 5.57. Pellets resolubilized in 8 M urea were fractionated on DEAE-Sephadex and analyzed by doubleNOnfasting were to immunodiffusion, SDS-PAGE, immunoelectrophoresis, and radial clot at 4OC. Following centrifugation, the serum was removed, immunodiffusion. IgG constituted 55-56% of the eluted protein. aliquoted into plastic tubes (Falcon 2054), and stored for varying When serum from all donors was fractionated by Staph A-Seph-periods at -70°C. Samples were obtained with informed consent. arose into IgG and non-IgG fractions, 85-89% of heparin precipi-The protocol was approved by the Human Experimentation Comtable protein was in the IgG fraction. mittee of University Hospitals. Subjects. The patients in this study were diagnosed as having C F by clinical criteria and elevated sweat chloride concentrations. Speculation Severity of disease was graded using the clinical scoring method We conclude from this study that the major heparin-precipitable outlined by Doershuk, et (3). For this study, patients with protein in human serum at pH 5.57 is l g~. The increased amounts scores over 75 are considered...

The Carbohydrate Convent of IgG from Patients with Cystic Fibrosis

“…Pearson and Lubin (29) reported that heparin precipitated more protein from CF serum than from normal serum and that the carbohydrate composition of the heparin-precipitated glycoproteins from CF sera contained 27% less sialic acid, 27% more fucose, and 3 1 % less hexose, despite the fact that total serum glycoproteins from both groups had the same carbohydrate content (30). We later showed that the major protein precipitated by heparin from serum is IgG, and that the amount of protein precipitated is directly related to the amount of IgG found in serum samples (24). Altered carbohydrate content probably has no relationship to increased heparin precipitation.…”

“…Abbreviations CF, cystic fibrosis SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis Several reports suggest that glycoproteins with N-glycosidically linked oligosaccharides from CF patients have altered carbohydrate content (1-3, 5, 6, 16, 22, 32). Pursuing the suggestion of Pearson and Lubin (30) that fucose, sialic acid, and hexose content of heparin-precipitated glycoproteins from serum of patients with CF are altered, we noted that IgG is the major serum component precipitated (24). In light of this finding, we have purified IgG from serum samples taken from healthy individuals, from patients with non-CF related pulmonary disease, and from CF patients and compared their carbohydrate contents.…”