Megalin binds and mediates cellular internalization of folate binding protein

Folate is an essential vitamin involved in a number of biological processes. High affinity folate binding proteins (FBPs) exist both as glycosylphosphatidylinositol‐linked, membrane associated folate binding proteins and as soluble FBPs in plasma and some secretory fluids such as milk, saliva and semen. The function and significance of FBPs are unresolved, however, it has been suggested that they may facilitate folate uptake, e.g. during suckling. The present study shows that megalin, a large, multiligand endocytic receptor and member of the low‐density lipoprotein‐receptor family, is able to bind and mediate cellular uptake of FBP. Surface plasmon resonance analysis shows binding of bovine and human milk FBP to immobilized megalin, but not to low density lipoprotein receptor related protein. Binding of 125I‐labeled folate binding protein (FBP) to sections of kidney proximal tubule, known to express high levels of megalin, is inhibitable by excess unlabeled FBP and by receptor associated protein, a known inhibitor of binding to megalin. Immortalized rat yolk sac cells, representing an established model for studying megalin‐mediated uptake, reveal 125I‐labeled FBP uptake which is inhibited by receptor associated protein and by antimegalin antibodies. Microinjection of 125I‐labeled FBP into renal tubules in vivo shows proximal tubular uptake by endocytosis. Megalin is expressed in several absorptive epithelia, including intestine and kidney proximal tubule, and thus the present findings provide a mechanism for intestinal and renal endocytic uptake of soluble FBP.

[1]  J. Sabourin,et al.  Comparative immunochemistry and ontogeny of two closely related coated pit proteins. The 280-kd target of teratogenic antibodies and the 330-kd target of nephritogenic antibodies. , 1993, The American journal of pathology.

[2]  J. E. Ford,et al.  Isolation of the folate‐binding protein from cow's milk by the use of affinity chromatography , 1972, FEBS letters.

[3]  J. Selhub,et al.  Milk folate binding protein (FBP): A secretory protein for folate? , 1984 .

[4]  M. Farquhar,et al.  Complete cloning and sequencing of rat gp330/"megalin," a distinctive member of the low density lipoprotein receptor gene family. , 1994, Proceedings of the National Academy of Sciences of the United States of America.

[5]  T. Hammond,et al.  The Intrinsic Factor-Vitamin B12 Receptor and Target of Teratogenic Antibodies Is a Megalin-binding Peripheral Membrane Protein with Homology to Developmental Proteins* , 1998, The Journal of Biological Chemistry.

[6]  D. N. Salter,et al.  Neonatal role of milk folate-binding protein: studies on the course of digestion of goat's milk folate binder in the 6-d-old kid , 1983, British Journal of Nutrition.

[7]  F. Greenwood,et al.  THE PREPARATION OF I-131-LABELLED HUMAN GROWTH HORMONE OF HIGH SPECIFIC RADIOACTIVITY. , 1963, The Biochemical journal.

[8]  J. Selhub,et al.  Renal folate absorption and the kidney folate binding protein. I. Urinary clearance studies. , 1987, The American journal of physiology.

[9]  J. Hjelle,et al.  Cell fractionation and electron microscope studies of kidney folate-binding protein. , 1991, The American journal of physiology.

[10]  E. Christensen,et al.  Internalization and intracellular transport of folate-binding protein in rat kidney proximal tubule. , 1993, The American journal of physiology.

[11]  J. Holm,et al.  Conversion of an apparent 100 kDa folate binding protein from human milk, choroid plexus and semen to a 25 kDa molecular species by phosphatidylinositol-specific phospholipase C , 1992, Bioscience reports.

[12]  S. Moestrup,et al.  Evidence for an essential role of megalin in transepithelial transport of retinol. , 1999, Journal of the American Society of Nephrology : JASN.

[13]  J. Ghitis.  The folate binding in milk. , 1967, The American journal of clinical nutrition.

[14]  E. Christensen,et al.  Megalin and cubilin: multifunctional endocytic receptors , 2002, Nature Reviews Molecular Cell Biology.

[15]  N. Hettiarachchy,et al.  Detection of a milk factor that facilitates folate uptake by intestinal cells. , 1981, Science.

[16]  H Birn,et al.  Megalin and cubilin: synergistic endocytic receptors in renal proximal tubule. , 2001, American journal of physiology. Renal physiology.

[17]  J. Holm,et al.  The complete amino acid sequence of the folate-binding protein from cow's milk , 1984 .

[18]  J. E. Ford.  Some observations on the possible nutritional significance of vitamin B12- and folate-binding proteins in milk , 1974, British Journal of Nutrition.

[19]  J. Holm,et al.  Isolation and characterization of the folate-binding protein from cow's milk , 1979 .

[20]  J. Fyfe,et al.  Cubilin dysfunction causes abnormal metabolism of the steroid hormone 25(OH) vitamin D3 , 2001, Proceedings of the National Academy of Sciences of the United States of America.

[21]  J. Mason,et al.  Folate-binding protein and the absorption of folic acid in the small intestine of the suckling rat. , 1988, The American journal of clinical nutrition.

[22]  P. Verroust,et al.  Immunofunctional properties of a yolk sac epithelial cell line expressing two proteins gp280 and gp330 of the intermicrovillar area of proximal tubule cells: inhibition of endocytosis by the specific antibodies. , 1995, European journal of cell biology.

[23]  J. Holm,et al.  Amino acid sequence homology between human and bovine low molecular weight folate binding protein isolated from milk , 1982 .

[24]  J. Holm,et al.  A Combination of Cation Exchange and Ligand-Affinity Chromatography for Purification of Two Molecular Species of the Folate Binding Protein in Human Milk, One Equipped with a Hydrophobic Glycosyl Phosphatidylinositol Tail: Characterization of Hydrophobicity and Electrical Charge , 2002, Bioscience reports.

[25]  R. Finnell,et al.  Renal tubular reabsorption of folate mediated by folate binding protein 1. , 2005, Journal of the American Society of Nephrology : JASN.

[26]  S. Nielsen,et al.  Internalization and apical-to-basolateral transport of folate in rat kidney proximal tubule. , 1997, The American journal of physiology.

[27]  S. Moestrup,et al.  Epithelial glycoprotein-330 mediates endocytosis of plasminogen activator-plasminogen activator inhibitor type-1 complexes. , 1993, The Journal of biological chemistry.

[28]  Henrik Vorum,et al.  An Endocytic Pathway Essential for Renal Uptake and Activation of the Steroid 25-(OH) Vitamin D3 , 1999, Cell.

[29]  S. Moestrup,et al.  Characterization of an Epithelial ∼460-kDa Protein That Facilitates Endocytosis of Intrinsic Factor-Vitamin B12 and Binds Receptor-associated Protein* , 1997, The Journal of Biological Chemistry.

[30]  J. Holm,et al.  High-affinity binding of folate to a protein in serum of male subjects. , 1980, Clinica chimica acta; international journal of clinical chemistry.

[31]  T. Willnow,et al.  Essential role of megalin in renal proximal tubule for vitamin homeostasis. , 1999, Journal of the American Society of Nephrology : JASN.

[32]  S. Moestrup,et al.  The human alpha 2-macroglobulin receptor contains high affinity calcium binding sites important for receptor conformation and ligand recognition. , 1990, Journal of Biological Chemistry.

[33]  A. Antony,et al.  Folate receptors. , 1996, Annual review of nutrition.

[34]  J. Holm,et al.  A high-affinity folate binding protein in human semen , 1991, Bioscience reports.

[35]  B. Seetharam,et al.  Cubilin and megalin expression and their interaction in the rat intestine: effect of thyroidectomy. , 2001, American journal of physiology. Endocrinology and metabolism.

[36]  C. Wagner.  Folate-binding proteins. , 1985, Nutrition reviews.

[37]  D. N. Salter,et al.  Influence of goat's-milk folate-binding protein on transport of 5-methyltetrahydrofolate in neonatal-goat small intestinal brush-border-membrane vesicles , 1988, British Journal of Nutrition.

[38]  S. Moestrup,et al.  Megalin-mediated endocytosis of transcobalamin-vitamin-B12 complexes suggests a role of the receptor in vitamin-B12 homeostasis. , 1996, Proceedings of the National Academy of Sciences of the United States of America.

[39]  T. Willnow,et al.  Lipoprotein receptors: new roles for ancient proteins , 1999, Nature Cell Biology.

[40]  D. Goldman,et al.  Membrane transport of folates. , 2003, Vitamins and hormones.