Characterization of soluble folate receptors (folate binding proteins) in humans. Biological roles and clinical potentials in infection and malignancy

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Abstract

This review surveys soluble Folate Receptors (FOLRs) in humans. FOLR1 and FOLR2 are equipped with cellular glycosylphosphatidylinositol (GPI) anchors. FOLR1 is secreted from epithelia with or without a micelle-encapsulated GPI-anchor into milk and other body fluids/secretions, e.g. semen where its interaction with spermatozoa indicates a role in male fertility. FOLR1 and FOLR2 serve as serum biomarkers of various diseases. FOLR3 possesses no GPI-anchor and originates from secretory granules of neutrophil granulocytes; its concentration in serum correlates to the FOLR3 content in leukocytes and rises with increased leukocyte counts (infection, malignancy and pregnancy). FOLR3 exerts anti-microbial and anti-tumor effects by depriving bacteria and tumor cells of natural folates. Megalin receptors mediate reabsorption of ultrafiltered folate-bound FOLR into cells of proximal kidney tubules and of folate-bound FOLR uptake in growing embryos. Megalin receptors overexpressed in malignant tumors could be suitable therapeutic targets for folate-conjugated cytotoxic agents utilizing soluble FOLRs as vectors.

Introduction

The high-affinity folate binding protein (FBP) plays a key role in the overall homeostasis of the vitamin folic acid in humans. One can distinguish between a membrane associated FBP which serves as a folate receptor (FR) attached to the outer cell surface by a glycosylphosphatidylinositol (GPI)-anchor, and a soluble form present in plasma and most body fluids [1]. The FR exists in two isoforms, FR α (FOLR1) and FR β (FOLR2). FOLR1 is expressed at the apical surface of normal epithelial tissues, and is overexpressed in several tumor cells where it has become a target for diagnostic imaging and cancer therapeutics [1,2]. FOLR2 is expressed in placental, hematopoietic cells and macrophages [1,2]. A third isoform, termed FR gamma (FOLR3) is secreted from myeloid cells as a soluble protein not possessing a GPI anchor [1,2].

The pathway for cellular internalization of the receptor is complex. The ligand-tagged FR is endocytosed by a non-clathrin and non-caveolar pathway into endosomes via GPI-anchored protein enriched endocytic compartments (GEECs) [2]. The biological function of FRs is to internalize folates into the cells, where the vitamin is of crucial importance to DNA synthesis and repair [1,2]. However, other important functions have recently been assigned to the receptor. Thus, it has been shown that the folate-FOLR1 complex mediates activation of the pro-oncogene STAT3 which contributes to tumor proliferation, angiogenesis and metastasis [3,4].

The presence of soluble forms of the high-affinity FR in milk, body fluids and plasma (serum) has been recognized over the past fifty years. In the earliest studies where neither the complete amino acid sequence nor the existence of different isoforms of FR was known, the concentration of soluble protein was determined indirectly as maximum high-affinity binding of radiolabeled folate (1–1 M stoichiometry). Hence, the concentration of protein was expressed as mass units of bound radiofolate (MW of folate 450 g). Recently the concentrations of soluble FOLR1 and FOLR2 in body fluids have been determined directly as mass concentrations (MW of protein 30,000 g) by immunological methods utilizing both poly- and monoclonal antibodies against FOLR1 and FOLR2.

The current literature has addressed the source of origin, identity (isoform) and nature of the soluble FR in plasma and body fluids, i.e. to what extent individual FR isoforms contribute to the total content of soluble FR in plasma and how physiological processes may regulate their concentrations. Data from recent and earlier studies have been compared disregarding that completely different analytical principles for measurement of soluble FR were applied in the early studies, i.e. mass units referring to bound radiofolate were thus compared to mass units referring to protein [5]. This has caused serious misconceptions and erroneous conclusions as to the identities and origins of the different soluble forms of FR in plasma or serum. The purpose of this review is therefore to address and analyze these topics critically and present a detailed and complete survey of the nature and function of soluble FR isoforms in plasma, body fluids-and secretions. Another issue to be discussed is whether soluble FOLR could serve as a candidate vector for folate conjugates of cytotoxic agents intended to target overexpressed megalin receptors in malignant tissues.

Section snippets

Secretion of soluble membrane-free FOLR1 with or without a retained GPI-anchor into body fluids

GPI-anchored proteins, like the folate receptors FOLR1 and FOLR2, belong to a group of proteins that are attached to the outer cell surface by a GPI-anchor. Their existence was established thirty years ago. They can be released from the intact cell surface into extracellular fluids as free water- soluble proteins devoid of a GPI-anchor after cleavage of the hydrophobic GPI-anchor. Two types of GPI-specific phospholipases in humans cleave GPI on different sites, GPI-phospholipase C and

FOLR isoforms in plasma or serum

The first studies reporting the presence of a high-affinity FBP in plasma/serum appeared more than 40 years ago. The concentration of FBP was determined by titration with tritiated folate to maximum binding. No definite conclusions as to origin, identity and function of the FBP were drawn at that time. The presence of soluble FOLRs in plasma/serum has recently attracted renewed attention after introduction of immunoassays based on antibodies specifically directed against FOLR1 and FOLR2. These

Soluble FOLR in granulocytes

The above evidence indicates that the high-affinity soluble FOLR in serum originates from the granulocyte. Hence, the molecular characteristics, identity and function of the FOLR isoform in neutrophil granulocytes will be addressed.

Distribution, homeostasis and cellular entry of soluble FOLR

The processes involved in distribution and homeostasis of soluble FOLRs, i.e. FOLR3, in humans have only been addressed in very few studies and are by far elucidated. However, evidence achieved in animal studies performed with bovine FBP indicates that the 600 kDa multiligand endocytic receptor megalin could play a crucial role [70]. Megalin binds a great number of structurally and functionally different ligands including several vitamin carrier proteins. Megalin is expressed in many

Conclusion and perspectives

The present review has analyzed the origin, presence and characteristics of the three soluble isoforms of FR in humans with a special focus on their biological functions and putative diagnostic or therapeutic potentials. Studies on high-affinity FOLRs in milk, other body fluids or secretions as well as plasma/serum have appeared over the past 50 years. The earliest studies used high-affinity binding of radiofolate to identify and determine the concentration of soluble FOLR in serum, whereas the

Declaration of Competing Interest

None.

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