Review article
Fatty acid binding protein isoforms: structure and function

https://doi.org/10.1016/S0009-3084(98)00003-6Get rights and content

Abstract

Although structural aspects of cytosolic fatty acid binding proteins (FABPs) in mammalian tissues are now well understood, significant advances regarding the physiological function(s) of these proteins have been slow in forthcoming. Part of the difficulty lies in the complexity of the multigene FABP family with nearly twenty identified members. Furthermore, isoelectric focusing and ion exchange chromatography operationally resolve many of the mammalian native FABPs into putative isoforms. However, a more classical biochemical definition of an isoform, i.e. proteins differing by a single amino acid, suggests that the operational definition is too broad. Because at least one putative heart H-FABP isoform, the mammary derived growth inhibitor, was an artifact (Specht et al. (1996) J. Biol. Chem. 271: 1943–49), the ensuing skepticism and confusion cast doubt on the existence of FABP isoforms in general. Yet, increasing data suggest that several FABPs, e.g. human intestinal I-FABP, bovine and mouse heart H-FABP, rabbit myelin P2 protein and bovine liver L-FABP may exist as true isoforms. In contrast, the rat liver L-FABP putative isoforms may actually be due either to bound ligand, post-translational S-thiolation and/or structural conformers. In any case, almost nothing is known regarding possible functions of either the true or putative isoforms in vitro or in vivo. The objective of this article is to critically evaluate which FABPs form biochemically defined or true isoforms versus FABPs that form additional forms, operationally defined as isoforms. In addition, recent developments in the molecular basis for FABP true isoform formation, the processes leading to additional operationally defined putative isoforms and insights into potential function(s) of this unusual aspect of FABP heterogeneity will be examined.

Introduction

As early as 20 years ago it was reported that rat and bovine liver fatty acid binding protein L-FABP could be separated into three putative isoforms by isoelectric focusing (Ketterer et al., 1976). During the ensuing two decades, considerable effort was expended in identifying the primary structure of these operationally defined isoforms, especially for L-FABP and in extending these observations to other FABPs. Four factors have hampered efforts to distinguish the structure and physiological function of these isoforms.

An operational definition of an L-FABP isoform is that the purified protein be resolvable into multiple forms, usually by isoelectric focusing or ion exchange chromatography. An operationally identified isoform would potentially include contaminating proteins unrelated to FABPs as well as FABP forms differing in amino acid composition, post-translational modification, conformation, bound ligand, or even ampholyte interactions (Ketterer et al., 1976). There is some evidence that the pI observed for specific FABP isoforms may also shift and/or arise from interactions of the FABP with the isoelectric focusing ampholyte. For example, delipidized adipocyte A-FABP species were interconvertible by nonequilibrium pH gel electrophoresis (Matarese et al., 1989). Similarly, differences in pIs of delipidized rat L-FABP isoforms, noted after loading the isoforms at the basic end of the gel (Frolov et al., 1997), were abolished by isoelectric focusing after loading the isoforms at the acidic end of the gel2. In contrast to the operational definition, the more strict and exclusive biochemical definition of a true isoform is a protein variant produced either as a result of different genes or from differential processing of singe gene transcripts. The resultant protein variants are true isoforms differing by a single amino acid at a single position in the peptide chain. While data on single amino acid substitutions has been presented for some FABP isoforms, convincing evidence demonstrating that they arise due to different genes or to differential RNA processing has to date been available only for the human I-FABP (Baier et al., 1995, Baier et al., 1996).

There are many different fatty acid/fatty acyl CoA binding proteins found in mammalian cytosol: nearly 20 FABPs (at least 6 sequenced and shown to be unique) (Matarese et al., 1989, Paulussen and Veerkamp, 1990, Banaszak et al., 1994, Borchers and Spener, 1994, Gossett et al., 1996); sterol carrier protein-2 (SCP-2) (Schroeder et al., 1995, Frolov et al., 1996); and acyl CoA binding protein (ACBP) (Gossett et al., 1996). All three protein families are found in high amount in cells: 3–5; 0.1–0.4; and 0.1–0.9% of cytosolic proteins are FABPs, SCP-2 and ACBP, respectively (Gossett et al., 1996). Furthermore, all three protein families colocalize, but differ somewhat in intracellular distribution, in enterocytes (L-FABP, I-FABP, SCP-2, ACBP), hepatocytes (L-FABP, SCP-2, ACBP) and heart myocytes (H-FABP, SCP-2, ACBP) (Gossett et al., 1996). There is some controversy whether SCP-2 is exclusively peroxisomal or also found in cytoplasm and other intracellular organelles (Van der Krift et al., 1985, Keller et al., 1989, van Heusden et al., 1990, Reinhart et al., 1993, Ossendorp et al., 1994, Schroeder et al., 1998).

This was dramatically demonstrated when ACBP was discovered copurifying with liver L-FABP (Knudsen, 1990, Gossett et al., 1996). The problem is further exacerbated in tissues such as the intestinal enterocyte which contains at least six different types of fatty acid/fatty acyl CoA bindings proteins in the 10–15 kDa molecular weight range: L-FABP, I-FABP (Paulussen and Veerkamp, 1990, ILBP (Miller and Cistola, 1993), I15P (Kanda et al., 1991, Fujii et al., 1993), ACBP (Gossett et al., 1996, Gossett et al., 1997) and SCP-2 Ossendorp et al., 1990, He et al., 1991, Moncecchi et al., 1991, Yamamoto et al., 1991, Seedorf et al., 1993, Ohba et al., 1994). None of the other proteins fit the biochemical definition of an FABP isoform. Yet, if these proteins copurify with native FABPs, then isoelectric focusing and/or ion exchange chromatography may operationally resolve them into erroneously identified FABP isoforms. Consequently, it is difficult to evaluate the older literature regarding FABP isoforms. Great care must be taken to assure that the FABP preparations are devoid not only of impurities but also of other copurifying fatty acid/fatty acyl CoA binding proteins.

Increasing data indicates that the amount of bound fatty acid, post-translational modification (phosphorylation, cysteinylation, glutathionylation, as well conformation alter the isoelectric focusing/ion exchange chromatographic behavior of an FABP. These alternate FABP forms are not true isoforms differing by a single amino acid.

Resolution of these issues is important to the understanding of the physiological function of both true isoforms and alternate FABP forms. For example, the majority of cellular unesterified fatty acid and fatty acyl CoA in mammalian tissues is associated with intracellular membranes (Gossett et al., 1996). However, the proportions found in the cytoplasm are dependent on the presence of fatty acid/fatty acyl CoA binding proteins (Knudsen, 1991, Faergeman and Knudsen, 1997, Jolly et al., 1997a). Within the cytoplasm, the unesterified fatty acids are not free, but instead more than 99% are bound to small cytosolic proteins, e.g. 14–16 kDa, FABPs (Glatz et al., 1993) and/or 13 kDa sterol carrier protein-2 (Schroeder et al., 1995, Stolowich et al., 1997). Likewise, cytoplasmic fatty acyl CoAs also appear largely bound to proteins, e.g. 10 kDa acyl CoA binding protein (ACBP) (Faergeman et al., 1996, Gossett et al., 1996), sterol carrier protein-2 (Frolov et al., 1996), or 14–16 kDa FABPs (Hubbell et al., 1994, Frolov and Schroeder, 1997, Frolov et al., 1997). What is the relative role of FABP isoforms and additional operationally defined forms in cellular fatty acid uptake, in cytosolic binding of fatty acids/fatty acyl CoAs, in intracellular trafficking/targeting of these ligands and in regulating fatty acid metabolism? Only recently, have these questions begun to be explored. A primary aim of this review is to detail some of these new findings.

Section snippets

Do all FABPs form operationally defined isoforms?

True FABP isoforms generally cannot be separated by gel permeation chromatography. Instead, in the majority of cases they have been operationally defined based on difference in charge, pI, and by resolution on isoelectric focusing or ion exchange chromatography. Over the past 20 years L-FABP (rat, bovine and human) has been separated into 2–4 putative isoforms by isoelectric focusing (Ketterer et al., 1976, Miyozawa and Hashimoto, 1979, Haunerland et al., 1984, Bass, 1985, Das et al., 1989,

Subclassification of operationally defined FABP isoforms

Not all of the operationally defined isoforms are true isoforms. In the following sections, the operationally defined FABP isoforms will be separated into four categories: (1) the true isoforms fitting the biochemical definition as protein forms differing by a single amino acid (Table 2); (2) the post-translationally modified forms (Table 3); (3) the conformational forms (Table 3); and (4) forms simply due to the amount of bound ligand, i.e. fatty acid (Table 1, Table 4). For the purposes of

Physiological function of FABP isoforms or alternate forms

With some exceptions shown below, little is known of the true function of FABP isoforms/alternate forms either in vitro or in vivo. Thus, this section of the review must be viewed with caution.

Conclusions and future directions

Although the early literature of operationally defined FABP isoforms is not conclusive with regard to whether these are biochemically defined isoforms (i.e. amino acid substitutions), increasing data indicate that several of the FABPs, especially H-FABP, are true isoforms. Most of the remaining operationally defined isoforms are not true isoforms, but instead appear due to bound ligands, post-translational modifications, or conformers. The latter brings up an interesting point—How would the

Acknowledgements

This work was supported by a grant from the USPHS NIH No. DK41402.

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