Afamin is a novel human vitamin E-binding glycoprotein characterization and in vitro expression

J Proteome Res. May-Jun 2005;4(3):889-99. doi: 10.1021/pr0500105.

Abstract

Hydrophobic vitamins are transported in human plasma and extravascular fluids by carrier proteins. No specific protein has been described so far for vitamin E, which plays a crucial role in protecting against oxidative damage and disease. We report here the purification of a 75-kDa glycoprotein with vitamin E-binding properties by stepwise chromatography of lipoprotein-depleted human plasma and monitoring of vitamin E (alpha-tocopherol)-binding activity. Partial sequencing identified this protein as afamin, a previously described member of the albumin gene family with four or five potential N-glycosylation sites. Glycosylation analysis indicated that >90% of the glycans were sialylated biantennary complex structures. The vitamin E-binding properties were confirmed using recombinantly expressed afamin. Qualitative and quantitative analysis of plasma and extravascular fluids revealed an abundant presence of this protein not only in plasma (59.8+/-13.3 microg/mL) but also in extravascular fluids such as follicular (34.4+/-12.7 microg/mL) and cerebrospinal (0.28+/-0.16 microg/mL) fluids, suggesting potential roles for afamin in fertility and neuroprotection. Afamin is partly (13%) bound to plasma lipoproteins. Afamin and vitamin E concentrations significantly correlate in follicular and cerebrospinal fluids but not in plasma. The vitamin E association of afamin in follicular fluid was directly demonstrated by gel filtration chromatography and immunoprecipitation which complements the in vitro findings for purified native and recombinant afamin.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Biological Transport
  • Body Fluids / chemistry
  • Carrier Proteins / chemistry
  • Carrier Proteins / isolation & purification
  • Carrier Proteins / metabolism*
  • Chromatography
  • Glycoproteins / chemistry
  • Glycoproteins / isolation & purification
  • Glycoproteins / metabolism*
  • Glycosylation
  • Humans
  • Immunoprecipitation
  • Lipoproteins / metabolism
  • Protein Binding
  • Serum Albumin / chemistry
  • Serum Albumin / isolation & purification
  • Serum Albumin / metabolism*
  • Serum Albumin, Human
  • Vitamin E / metabolism*

Substances

  • AFM protein, human
  • Carrier Proteins
  • Glycoproteins
  • Lipoproteins
  • Serum Albumin
  • Vitamin E
  • Serum Albumin, Human