The Menagerie of Human Lipocalins: A Natural Protein Scaffold for

Article pubs.acs.org/accounts

The Menagerie of Human Lipocalins: A Natural Protein Scaffold for Molecular Recognition of Physiological Compounds André Schiefner and Arne Skerra* Munich Center for Integrated Protein Science (CIPS-M) and Lehrstuhl für Biologische Chemie, Technische Universität München, 85350 Freising-Weihenstephan, Germany CONSPECTUS: While immunoglobulins are well-known for their characteristic ability to bind macromolecular antigens (i.e., as antibodies during an immune response), the lipocalins constitute a family of proteins whose role is the complexation of small molecules for various physiological processes. In fact, a number of low-molecular-weight substances in multicellular organisms show poor solubility, are prone to chemical decomposition, or play a pathophysiological role and thus require specific binding proteins for transport through body fluids, storage, or sequestration. In many cases, lipocalins are involved in such tasks. Lipocalins are small, usually monomeric proteins with 150−180 residues and diameters of approximately 40 Å, adopting a compact fold that is dominated by a central eight-stranded up-and-down β-barrel. At the amino-terminal end, this core is flanked by a coiled polypeptide segment, while its carboxy-terminal end is followed by an α-helix that leans against the β-barrel as well as an amino acid stretch in a more-or-less extended conformation, which finally is fixed by a disulfide bond. Within the β-barrel, the antiparallel strands (designated A to H) are arranged in a (+1)7 topology and wind around a central axis in a right-handed manner such that part of strand A is hydrogen-bonded to strand H again. Whereas the lower region of the β-barrel is closed by short loops and densely packed hydrophobic side chains, including many aromatic residues, the upper end is usually open to solvent. There, four long loops, each connecting one pair of β-strands, together form the entrance to a cupshaped cavity. Depending on the individual structure of a lipocalin, and especially on the lengths and amino acid sequences of its four loops, this pocket can accommodate chemical ligands of various sizes and shapes, including lipids, steroids, and other chemical hormones as well as secondary metabolites such as vitamins, cofactors, or odorants. While lipocalins are ubiquitous in all higher organisms, physiologically important members of this family have long been known in the human body, for example with the plasma retinol-binding protein that serves for the transport of vitamin A. This prototypic human lipocalin was the first for which a crystal structure was solved. Notably, several other lipocalins were discovered and assigned to this protein class before the term itself became familiar, which explains their diverse names in the scientific literature. To date, up to 15 distinct members of the lipocalin family have been characterized in humans, and during the last two decades the three-dimensional structures of a dozen major subtypes have been elucidated. This Account presents a comprehensive overview of the human lipocalins, revealing common structural principles but also deviations that explain individual functional features. Taking advantage of modern methods for combinatorial protein design, lipocalins have also been employed as scaffolds for the construction of artifical binding proteins with novel ligand specificities, so-called Anticalins, hence opening perspectives as a new class of biopharmaceuticals for medical therapy. the strong conservation of the β-barrel with the attached αhelix, the sequence similarity among this class of proteins was extremely low, often just around 10% residue identity. On the basis of this knowledge, many more proteins with known amino acid sequence were assigned to the lipocalin family, even in the absence of high-resolution structural information. Moreover, it was discovered that there are similarities in the intron/exon structure of the genes for eukaryotic lipocalins,5 which allowed the identification of further family members. Today, the UniProt database (http://www.uniprot.org) reveals close to 1000 lipocalins, among which are 38 entries for human lipocalins (Table 1), which however includes several isoforms as well as more

1. INTRODUCTION The term lipocalin was coined 30 years ago for a group of proteins comprising the human α1-acid glycoprotein, α1microglobulin (also known as protein HC), serum/plasma retinol-binding protein, and bovine β-lactoglobulin on the basis of sequence homologies, disulfide bond pattern, and functional similarities, reflecting their ability to complex lipophilic molecules inside a cup-shaped (Greek calyx) protein fold.1 At this time, the crystal structure of just one human member of this family had been elucidated, the retinol-binding protein,2 which remained the only human representative with known three-dimensional (3D) structure until 2000.3 Through a comparison with the published crystal structures of other lipocalin proteins from animal species, three main structurally conserved regions (SCRs 1−3) in the lipocalin fold were identified.4 From this analysis it became clear that despite © XXXX American Chemical Society

Received: November 21, 2014

A

DOI: 10.1021/ar5003973 Acc. Chem. Res. XXXX, XXX, XXX−XXX

B

a

168

prostaglandin D/H2

fatty acids, fatty alcohols, cholesterol, glyco- and phospholipids

lachrymal gland, salivary gland

158

155

155

164 178

unknown iron-complexed catecholate-type ferric siderophore, enterobactin, bacillibactin, L-norepinephrine vanillin, lilial, linear aldehydes, fatty acids, branched alcohols, etc. broad ligand spectrum, cf. OBPIIa

162

sperm head, retinoic acid

182

188

184 169

183

M

M

Mc

M

M M

D

H

M/HDL

M M/H/HDL

M

M

M

≤183 183

oligomeric statea

no. of residues

nasal mucosa, lachrymal and salivary glands prostate, mammary gland CNS, heart

endometrium, fallopian tube, seminal gland duodenumb neutrophils

spingosine-1-phosphate, myristic acid, glycerol myristate C8α, lauric acid

heme, 3-hydroxykynurenine progesterone, arachidonate

disopyramide, amitriptyline, chlorpromazine

all-trans retinol, 2-(4-(2-(trifluoromethyl)phenyl) piperidine-1-carboxyamino)benzoic acid warfarin, 7-hydroxystaurosporine, imatinib

physiological/investigated ligands

P07360 P09466 Q6UWW0 P80188

Q9NY56

− N28, N63 − N65 −

C40 − C26 C87

66−160, 106−119 63−156c 76−175

61−153

67−164

59−151c

59−151

76−168

C101

C43, C145

C99

C99

P41222

P31025

−

Q9NPH6

P02760 P05090

P19652

S7, N29, N56

−

O95445

N135

C34 C116

C149

−

23−167, 95−183, 128−157

72−169 8−114, 41−165

5−147, 72−165

P02763

N15, N38, N54, N75, N85 N15, N38, N54, N75, N85 T5, N17, N96 N45, N78

5−147, 72−165

P02753

UniProt ID

(C149)d

glycosylation sites −

free Cys −

4−160, 70−174, 120−129

disulfide bridges

M, monomer; D, dimer; H, heterodimer via intermolecular disulfide bond; HDL, HDL-associated. bUncertain. cBy similarity. dORM1 gene polymorphism: p.Arg167Cys.

lipocalin-15 (Lcn15) neutrophil gelatinaseassociated lipocalin (NGAL) odorant-binding protein IIa (OBPIIa) odorant-binding protein IIb (OBPIIb) lipocalin-type prostaglandin-D synthase (PGDS) tear lipocalin (Tlc)

complement component C8 γ chain (C8γ) glycodelin (Gd)

liver

liver CNS, adrenals, spleen, placenta liver, kidney

α1-microglobulin (A1M) apolipoprotein D (ApoD)

apolipoprotein M (ApoM)

liver

liver

liver

tissue of primary synthesis

α1-acid glycoprotein 2 (AGP2)

plasma retinol-binding protein (RBP) α1-acid glycoprotein 1 (AGP1)

lipocalin (abbreviation)

Table 1. Overview of Human Lipocalins

1XKI, 3EYC

90% identity to 4RUN 2WWP, 3O2Y, 4IMO, 4OS3

4RUN

2XST 1NGL, 1L6M, 1X8U, 3CMP

2WEW, 2WEX, 2YG2 1LF7, 2OVD, 2QOS, 3OJY 4R0B

3QKG, 4ES7 2HZQ, 2HZR

3APU, 3APV, 3APW, 3APX

1RBP, 1QAB, 3BSZ, 3FMZ 3KQ0

representative PDB IDs

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Accounts of Chemical Research

Figure 1. Gallery of human lipocalin structures. Representative crystal structures of human lipocalins are shown as cartoons (PDB IDs: 1RBP, 3KQ0, 3QKG, 2HZQ, 2WEW, 2QOS, 4R0B, 2XST, 1L6Ma, 4RUNa, 4OS3a, and 3EYCa). Because of the high structural similarity with AGP2, only the AGP1 isoform is depicted. Cys residues are highlighted as ball-and-stick: disulfide bridges (yellow), unpaired Cys (red), and catalytically active Cys (blue). Gray dashed residues of Lcn15 indicate a loop region not defined in the crystal structure.

remotely related “lipocalin-like” proteins. This intricate scientific genesis of the lipocalin family distinguishes these proteins from the immunoglobulin (super)family, for example, whose members share much higher similarity on the sequence level. The lipocalins form a diverse class of ligand-binding proteins that serve for the transport, storage, or sequestration of a variety of small molecules in most genera of life, from bacteria to man. In humans, the lipocalins constitute secretory proteins that occur in the blood plasma or other body fluids, including genital secretions and tears. After most of these proteins were cloned and made available in a recombinant form,6 the crystal structures of essentially all human representatives of the lipocalin family have been solved. Here we present the first review of the structures and functions of the 12 major human lipocalins (Figures 1 and 2).

hepatocytes, RBP picks up retinol in the endoplasmic reticulum prior to its secretion.10 In plasma, the RBP·retinol complex associates with homotetrameric transthyretin (prealbumin), thus preventing premature renal clearance.9 In the eye, binding to the STRA6 receptor causes a conformational change that triggers both release of retinol and dissociation of the transthyretin complex.11

3. OTHER LIPOCALINS IN HUMAN PLASMA α1-Acid glycoprotein (AGP) represents one of the major acute phase proteins.12 AGP is mainly synthesized by hepatocytes and exists as a mixture of two subtypes, F1*S (AGP1) and A (AGP2), which differ by 20 residues.13 Both variants carry five N-linked glycans, which cause an exceptionally high carbohydrate content of 45% in total weight, while the high proportion of sialic acid results in an unusually low pI. Although the precise biological function of the two AGP subtypes is still unclear, their binding activity for numerous therapeutic compounds strongly affects the bioavailability of these drugs, also depending on the oligosaccharide composition, which influences ligand selectivity.14 The first X-ray structure of AGP was solved for the F1*S isoform with a small physiological molecule bound in the ligand pocket, which furthermore accommodated the N-terminal pyroglutamate residue of a

2. RETINOL-BINDING PROTEIN: THE PROTOTYPIC LIPOCALIN Retinol-binding protein (RBP; gene name RBP4) serves to transport vitamin A (retinol) in human plasma. Its deep and narrow hydrophobic pocket is perfectly molded to accommodate this hydrophobic and chemically sensitive ligand but can also complex non-retinoid compounds.7−9 Synthesized by C


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Figure 2. Structure-based amino acid sequence alignment for the human lipocalins. (A) The alignment was generated with SALIGN,77 and the loop regions with their highly diverse sequences were manually condensed to better match the 3D situation. Secondary structure elements are highlighted in green (β-strand) and light blue (α-helix). Except for ApoM, where # indicates 17 additional N-terminal residues, all of the sequences start with residue 1 of the mature polypeptide (Z denotes an N-terminal pyroglutamyl residue). The common GXW motif4 and the conserved set of 58 Cα positions (=),73 which are useful for pairwise superposition, are labeled below the alignment. Residues (mostly Cys) that had been replaced during crystallographic analysis were reverted according to the original sequence. Cys residues either involved in disulfide bonds, unpaired, or relevant for catalytic activity are highlighted in yellow, red, and blue, respectively. Known N- and O-glycosylation sites are colored violet. Structurally disordered residues are depicted in lower case. (B) Three-dimensional illustration of the structural superposition/alignment. The conserved β-strands are shown in black, the variable loops 1−4 are colored as in Figure 1, and the remaining segments are depicted in gray. D


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Accounts of Chemical Research neighboring molecule in the crystal.15 Subsequently, X-ray structures of AGP2 were elucidated in complex with the drugs amitriptyline, disopyramide, and chlorpromazine.16 α1-Microglobulin (A1M) is abundant in blood plasma and also in connective tissue and urine.17 Its biosynthesis in human hepatocytes is unique among the human lipocalins insofar as A1M shares its structural gene, termed α1m/bikunin precursor protein (AMBP), with the Kunitz-type serine proteinase inhibitor bikunin. In the plasma, ∼50% of A1M forms a covalent heterodimer with immunoglobulin A via its unpaired Cys residue C34.17 A1M has been associated with various immunological processes and also exhibits reductase and radical scavenging functions as well as cell-protection activities, relying on C34, three Lys residues at the entrance of the β-barrel, and cofactors such as heme, NADH, or ascorbate.18 Apart from that, A1M serves as a biomarker for renal tubular dysfunction and toxicity, preeclampsia, and also hepatitis E.19,20 Indeed, the first crystal structure of A1M provided evidence for a potential heme-binding site21 and was recently confirmed.22 Neutrophil gelatinase-associated lipocalin (NGAL, Lcn2), also called siderocalin, is constitutively expressed in neutrophil granules and secreted in response to activation of the innate immune system during bacterial infection.23 It acts as a bacterial growth inhibitor by scavenging certain siderophores, in particular enterobactin (enterochelin) from Escherichia coli, which are utilized by microbes to sequester ferric ions from human body fluids.24 The protein carries an unpaired Cys residue, C87, which is known to provoke the formation of homodimers or conjugates with gelatinase (MMP9). 25 Crystallographic and ligand binding studies revealed a rather shallow and wide ligand pocket24 that can accommodate a spectrum of catecholic or phenolic iron chelators (with dissociation constants in the subnanomolar range)26, but also iron-complexed L-norepinephrine.27 Another plasma lipocalin, C8γ, constitutes one of three subunits of the complement component C8, which assembles with four additional components to form the cytolytic membrane attack complex (MAC).28 C8γ is synthesized by hepatocytes and secreted as a disulfide-linked C8α/γ heterodimer, which non-covalently associates with C8β.29 Structural studies of C8γ revealed a deep cavity divided into a nonpolar lower part that can harbor slim hydrophobic ligands (e.g., lauric acid) and a solvent-exposed upper part that interacts with a β-hairpin loop of C8α.30−32 The intermolecular disulfide bridge to C164 of C8α is formed by the Cys residue C40 in loop no. 1 of C8γ.

and in complex with progesterone, another well-known physiological ligand.40 Apolipoprotein M (ApoM) is also found predominantly in association with HDL. In this case, the unprocessed hydrophobic N-terminal signal peptide anchors the protein in the phospholipid bilayer of the lipoprotein particle, thus utilizing a different mechanism than ApoD to prevent renal clearance.41 ApoM is expressed in hepatocytes and kidney proximal tubule cells.42,43 Studies of ApoM in mice demonstrated antiatherogenic effects, which are probably related to its role in cholesterol efflux from macrophage foam cells, the formation of pre-β-HDL, and also antioxidative activity.44,45 Furthermore, ApoM shows vasculoprotective effects in mice by delivering sphingosine-1-phosphate (S1P) to its receptor on endothelial cells.46 Crystal structures of ApoM in complex with myristic acid, glycerol-1-myristate, and S1P have been solved.46,47 Lipocalin-type prostaglandin D synthase (PGDS), also known as β-trace, is the major enzyme to catalyze the formation of prostaglandin D2 (PGD2) from PGH2 in the CNS, while it is also found in heart, prostate, and epididymis.48 Notably, Cys residue C43, whose thiol side chain points into the ligand pocket of this lipocalin, acts as the catalytic center. The PGDS concentration is correlated with neurological pathologies such as normal-pressure hydrocephalus, spinal canal stenosis, and subarachnoid hemorrhage.49 Furthermore, this enzymatic lipocalin can also bind a broad range of hydrophobic ligands, including bilirubin, biliverdin, retinoids, thyroids, steroids, and flavonoids.50 In accordance with this, structural studies have revealed a rather large substrate/ligand pocket for PGDS.51−53 Glycodelin (Gd) is involved in crucial biological processes such as reproduction and immune reaction.54 Four functionally distinct glycoforms of Gd have been identified in reproductive tissues and fluids: Gd-A, Gd-C, Gd-F, and Gd-S. Mature Gd has two N-glycosylation sites that can carry differing oligosaccharides.55,56 Notably, Gd is the only human lipocalin that lacks a pronounced central pocket for ligand binding inside the βbarrel. However, this lipocalin forms a structurally unique homodimer that serves as a scaffold for the simultaneous presentation of four large sugar side chains on the same face of the protein,57 thus strengthening receptor interactions via an avidity effect. Interestingly, Gd orthologues have been found only in those primate suborders that show a menstrual cycle. In contrast, the gene for the closest Gd homologue, βlactoglobulin, which represents an abundant lipocalin in other mammals, is deactivated in humans.58 Tear lipocalin (Tlc, Lcn1) is the major protein constituent in human tear fluid and is mainly expressed in the lachrymal and salivary glands, beside prostate, nasal, and tracheal mucosa.59 Tlc has been isolated from biological samples in complex with diverse endogenous ligands such as cholesterol, fatty acids and alcohols, and glyco- and phospholipids.60,61 The physiological role of Tlc appears to be preservation of the aqueous−lipid interface of the tear film as well as scavenging of both potentially harmful lipid degradation products and microbial siderophores.62 Cellular internalization of Tlc via the lipocalin-1 interacting membrane receptor (LIMR) may be involved in detoxification processes.63 Different crystal structures of Tlc have indicated elevated conformational flexibility for this lipocalin, especially in the loop region at the entrance to the ligand pocket.64,65 Odorant-binding protein (OBP) presumably acts as a carrier for airborne hydrophobic molecules, which must traverse the

4. LIPOCALINS WITH DISTINCT LOCALIZATION Apolipoprotein D (ApoD) forms part of the high-density lipoprotein (HDL) fraction in plasma33 but lacks the characteristic amphipathic α-helical structure of other apolipoproteins. Instead, ApoD peripherally associates with the HDL micelle via an intermolecular disulfide bridge between its Cys residue C116 and C6 in ApoA-II.34 Expression of ApoD is found in several tissues with the highest mRNA levels in adrenals, spleen, placenta, and, notably, the central nervous system (CNS).35 This lipocalin received attention as a prognostic marker for prostate cancer,36 breast carcinoma, and cutaneous malignant melanoma.37 In the CNS, ApoD is synthesized by astrocytes and seems to be involved in arachidonic acid transport and metabolism,38 also playing a role in psychiatric disorders such as schizophrenia.39 The 3D structure of ApoD was elucidated both in the ligand-free form E


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illustrates that the core of the human lipocalins comprises a strongly conserved conical eight-stranded β-barrel that is closed on one end and provides a hydrophobic ligand pocket at the opposite end, which is usually open to solvent (Figures 2 and 3). The four loops 1−4 connect the β-strands A−H in a

aqueous milieu of the nasal mucus before binding to cell-surface receptors.66 Two isoforms of OBP, sharing 90% residue identity, have been identified in humans: OBPIIa is expressed in nasal tissues, salivary gland, lachrymal glands, and lung, whereas OBPIIb is preferentially expressed in the prostate and mammary glands.67 Investigation of the ligand-binding spectrum of OBPIIa revealed a variety of chemically diverse odorants such as lilial, vanillin, linear aldehydes, fatty acids, and the like.68 With this ligand promiscuity OBP resembles Tlc; in fact, it constitutes its closest human paralogue, probably as result of a recent gene duplication event. In contrast to the dimeric bovine OBP66 and similar to other mammalian orthologues, human OBPIIa is a monomer, as its crystallographic analysis has recently revealed.69

5. ELUSIVE LIPOCALINS Lipocalin-15 (Lcn15) was identified in a screen for novel human secreted proteins.70 Lcn15 seems to be localized in the gastrointestinal tract (http://www.proteinatlas.org); however, no functional data are yet available. Its X-ray structure was solved in a ligand-free state (Muniz et al., to be published; PDB ID 2XST; cf. Figure 1). Lipocalin-6 (Lcn6) was discovered as a member of three closely related putative gene products, together with Lcn5 and Lcn8, by human genome analysis. Its gene (LCN6) was found on chromosome 9q34 adjacent to LCN5 (homologous to murine mE-RABP) and LCN8 (homologous to murine mEP17). Lcn6 was characterized as a novel human epididymal lipocalin located on the head and tail of spermatozoa, thus suggesting a role in male fertility.71 However, Lcn6 differs from all other human lipocalins as it contains neither a disulfide bridge nor a glycosylation site and, with only 143 residues, its mature size is much smaller. Inspection of the Lcn6 mRNA (GenBank accession code AF303084) indicates truncation of a longer reading frame by 17 residues (including the C-terminal Cys residue) due to a nonsense mutation. Notably, a full-length Lcn6 version is found in orangutan and gibbon, in which C61 and C154 seem to form the typical disulfide bridge between βstrand D and the C-terminus (see further below). This suggests that the nonsense mutation has occurred only recently during primate evolution in the Hominoidea family, somewhat reminiscent of the situation for β-lactoglobulin, whose gene is deactivated in the same evolutionary branch.57 Finally, comparison of human chromosome 9q34 with the murine chromosome 2A3 led to the identification of further putative epididymal lipocalins in humans: Lcn9, Lcn10, and Lcn12.72 Indeed, according to the human proteomics database (https://www.proteomicsdb.org), there is evidence for both Lcn8 (see above), Lcn9, and Lcn10 expression; furthermore, Lcn12 has been detected in human thyroid gland and other tissues by antibody staining (http://www.proteinatlas.org). However, nothing about their biological roles is known to date.

Figure 3. Space-filling representation of the lipocalin pocket shapes. All of the cavities are illustrated with a translucent surface based on Xray coordinate sets (PDB IDs: 1RBP, 3APX, 3QKG, 4R0B, 2XST, 3CMPa, 2HZQ, 2YG2b, 3OJY, 4RUNa, 4IMO, and 3EYCa) using colors analogous to Figure 1. As far as is evident from the crystal structures, ligands of the following complexes are depicted as sticks: RBP·retinol, AGP·chlorpromazine, NGAL·enterobactin, ApoD·progesterone, ApoM·sphingosine-1-phosphate, C8γ·C8α, PGDS·analogU44069 and Tlc·butanediol. The thiol group of the C8α peptide, which gives rise to a covalent cross-link for this ligand to C8γ, is highlighted as an orange sphere.

pairwise fashion and form the entrance of the cavity. The Cterminal α-helix, which leans against a concave region of the βbarrel (cf. Figure 1), is also positioned in a remarkably similar manner throughout all of the members. As proposed before,73 the β-barrel core can be described by a set of 58 structurally conserved Cα positions, which for the human representatives reveals pairwise root-mean-square deviations (RMSDs) of less than 2 Å relative to RBP (Table 2). In contrast, the four loops 1−4 not only considerably differ in their sequences and lengths (cf. Figure 2 and Table 2) but also exhibit substantial conformational plasticity. As result, the ligand pockets differ drastically in size (cf. Figure 3), ranging from a rudimentary

6. COMMON STRUCTURAL FEATURES OF THE HUMAN LIPOCALINS The availability of 3D structures has enabled for the first time a comprehensive structure-based sequence alignment for the major human lipocalins (Figure 2). All of these abundant human representatives exhibit the classical lipocalin fold (Figure 1), even though their amino acid sequences are highly diverse, sharing only two identical residues, i.e., Gly and Trp of the GXW motif within SCR1.4 Structural superposition F


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Coordinate sets were taken from PDB entries 1RBP, 3KQ0, 3APX, 3QKG, 2HZQ, 2WEW, 2QOS, 4R0B, 2XST, 1L6Ma, 4RUN, 4OS3a, and 3EYCa. RBP served as a reference for pairwise superposition and RMSD calculation using the structurally conserved 58 β-barrel residues (see the text). bThe number in parentheses is the number of residues in the loop.

(closed) cavity in Gd (54 Å3) to quite a large pocket in C8γ (2500 Å3). All of the human lipocalins with known tertiary structure, except for ApoD, carry a disulfide bridge that fixes the stretched C-terminus (β-strand I) to β-strand D of the β-barrel; in ApoD this link is formed to β-strand B (Figures 1 and 2). RBP, AGP1/2, ApoD, ApoM, and Gd exhibit additional disulfide bridges, which in RBP, AGP1/2, and ApoM fix the N-terminal coiled segment to the C-terminal end of the α-helix. A third disulfide bridge is unique to RBP and ApoM, where it connects β-strands G and H or the N-terminal end of the α-helix with the FG loop (at the bottom of the β-barrel), respectively. In Gd, the second disulfide cross-link also links β-strands G and H, but at different positions. Interestingly, the second disulfide bridge of ApoD is formed between the N-terminal segment and a Cys residue in β-strand G that is structurally equivalent to the one involved in the third disulfide cross-link of RBP. In contrast to many other extracellular proteins, in particular immunoglobulins, most of these disulfide bonds are not buried inside the hydrophobic core but instead are solvent-exposed; this suggests that they rather serve to fix the loose ends of the polypeptide chain, most likely to protect lipocalins from attack by exopeptidases in human body fluids. In addition, 10 of the human lipocalins exhibit one unpaired Cys side chain (Table 1), but without conserved location (Figures 1 and 2A). At least for A1M, ApoD, C8γ, and NGAL, this has been shown to evoke covalent heterodimer formation with other plasma proteins, resulting in retarded kidney filtration of the macromolecular complexes. Presumably, the unpaired Cys side chains fulfill similar functions in Lcn15, AGP, and PGDS. Only in the OBPs and Tlc, where the Cys residue is situated inside the β-barrel, the thiol group may be involved in ligand binding; likewise, one of the two unpaired thiol side chains of PGDS, as well as the one of A1M, is crucial for catalytic activity. ApoM and RBP, which both lack a free thiol group, achieve prolonged plasma half-lives via different mechanisms, i.e., via an N-terminal micelle-anchoring peptide and ligand-dependent complex formation with transthyretin, respectively. As a further option, AGP utilizes an increased size resulting from its extended multiple N-linked glycosylation to slow down renal excretion. While roughly half of the human lipocalins are glycoproteins, their glycosylation sites are scattered, mainly residing in the Nterminal half of the amino acid sequences (cf. Figure 2). Furthermore, the composition of the oligosaccharide side chains varies considerably, and in the case of Gd a differing glycosylation is even crucial for the biological function. In conclusion, the structural comparison of all well-characterized human lipocalins highlights the conformational plasticity of the loop region at the open end of the β-barrel as well as the versatility of the cavity (cf. Figures 2B and 3), which are both responsible for the diverse physiological roles in ligand binding. Overall, the lipocalin fold tolerates an enormous sequence variation among the human paralogues while maintaining a uniform β-barrel structure.

7. FROM NATURAL LIPOCALINS TO ANTICALINS The modular architecture of the lipocalins, with their set of four structurally hypervariable loops supported by a rigid β-barrel, in principle resembles that of the antigen-binding domains of antibodies, with their six complementarity-determining regions (CDRs). On the basis of this notion, lipocalins were employed as an alternative protein scaffold to engineer novel binding

a

121−131 116−121 116−121 118−126 115−123 138−141 122−125 109−116 106−113 125−132 101−108 113−121 103−110 89−101 (13) 91−96 (6) 91−96 (6) 91−98 (8) 88−95 (8) 114−118 (5) 95−102 (8) 85−89 (5) 82−86 (5) 95−105 (11) 78−81 (4) 86−93 (8) 80−83 (4) (12) (7) (7) (7) (8) (8) (7) (7) (7) (6) (7) (7) (7) 59−70 66−72 66−72 66−72 60−67 88−95 70−76 60−66 57−63 70−76 53−59 61−67 55−61 (10)b (15) (15) (15) (9) (15) (15) (16) (15) (14) (14) (15) (13) 31−40 32−46 32−46 32−46 33−41 54−68 38−52 26−41 24−38 38−51 21−34 28−42 24−36 − 1.97 1.79 0.98 0.86 1.52 1.19 1.38 1.18 0.99 1.40 1.01 1.52 132−138 122−128 122−128 127−133 124−130 142−148 126−132 117−123 114−120 133−139 109−115 122−128 111−117 114−120 109−115 109−115 111−117 108−114 131−137 115−121 102−108 99−105 118−124 94−100 106−112 96−102 102−109 97−104 97−104 99−106 96−103 119−126 103−110 90−97 87−94 106−113 82−89 94−101 84−91 85−88 87−90 87−90 87−90 84−87 110−113 91−94 81−84 78−81 91−94 74−77 82−85 76−79 52−58 59−65 59−65 59−65 53−59 81−87 63−69 53−59 50−56 63−69 46−52 54−60 48−54 RBPa AGP1 AGP2 A1M ApoD ApoM C8γ Gd Lcn15 NGAL OBPIIa PGDS Tlc

21−30 22−31 22−31 22−31 23−32 44−53 28−37 16−25 14−23 28−37 11−20 18−27 14−23

41−47 47−53 47−53 47−53 42−48 69−75 53−59 42−48 39−45 52−58 35−41 43−49 37−43

71−78 73−80 73−80 73−80 68−75 96−103 77−84 67−74 64−71 77−84 60−67 68−75 62−69

F E D C B A

strands of the β-barrel

Table 2. Definition of Conserved β-Barrel Segments and Hypervariable Loops

G

H

RMSD [Å]

no. 1 A−B

no. 2 C−D

loops

no. 3 E−F

no. 4 G−H

(11) (6) (6) (9) (9) (4) (4) (8) (8) (8) (8) (9) (8)


G


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Accounts of Chemical Research proteins, so-called Anticalins, via combinatorial protein design.73 In this regard, the human lipocalins have attracted special attention,74 as their abundance in the body implies high tolerance if administered as biological drugs. Anticalins have been successfully generated on the basis of human Tlc and NGAL (among others) in an in vitro process that fundamentally resembles the humoral immune response against an antigen.75 A predefined set of up to 20 amino acid positions distributed across the entire loop region was subjected to random mutagenesis followed by target-specific selection via phage display. In this manner, lipocalins were engineered to recognize prescribed small molecules of the immunological hapten type, similar to their biological counterparts; furthermore, Anticalins are also capable of tightly binding (with dissociation constants down to the pM range) peptides and, in particular, protein antigensboth soluble and as part of cell-surface receptorsthus covering the full spectrum of medically relevant targets (Figure 4).

Thus, the lipocalin scaffold appears promising to provide binding proteins against a wide range of target molecules with different sizes, shapes, and biomolecular properties. This opens prospects for medical applications by means of several therapeutic principles, such as (i) blocking soluble signaling factors or cellular receptors, (ii) scavenging toxic or irritating molecules, and (iii) delivering drugs including toxic payloads. Hence, it is likely that in the future, in a therapeutic setting, endogenous human lipocalins will be complemented with engineered counterparts, comparable to the application of recombinant monoclonal antibodies for immunotherapy.

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AUTHOR INFORMATION

Corresponding Author

*Address: Lehrstuhl für Biologische Chemie, Technische Universität Mü nchen, Emil-Erlenmeyer-Forum 5, 85350 Freising-Weihenstephan, Germany. Phone: +49 8161 71 4351. Fax: +49 8161 71 4352. E-mail: [email protected]. Author Contributions

Both authors contributed equally to this work. Notes

The authors declare the following competing financial interest(s): A.Sk. is founder and shareholder of Pieris Parmaceuticals, Inc., a biotech company that commercializes the Anticalin technology. Biographies André Schiefner is a senior research associate and group leader at Technische Universität München, Germany. He received his Ph.D. in Structural Biochemistry from the University of Konstanz, followed by postdoctoral studies at The Scripps Research Institute, La Jolla, CA. His work focuses on structural aspects of protein engineering, enzymatic catalysis, and treatment of human disease. Arne Skerra is Full Professor and holds the Chair of Biological Chemistry at Technische Universität München, Germany. He received his Ph.D. in Biochemistry from the Ludwig-Maximilians-Universität München. He completed a postdoctoral fellowship at the Laboratory of Molecular Biology in Cambrige, U.K. and was group leader at the Max-Planck-Institut für Biophysik in Frankfurt am Main, followed by a position as Associate Professor for Protein Chemistry at the Technische Universität Darmstadt, Germany. His research area encompasses protein design, structural analysis, and protein biochemistry.

Figure 4. Examples of engineered human lipocalins. The crystal structures of NGAL-based Anticalins in complex with different types of medically relevant target molecules are shown as cartoons (colored): Y·DTPA (ball-and-stick, gray; 3DSZa), amyloid-β peptide (ball-andstick, gray; 4MVLa), and CTLA-4 (cartoon, gray; 3BX7). Cα positions of mutated residues in each binding site are highlighted as green spheres.

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REFERENCES

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An Anticalin directed against the hepcidin peptide, a negative regulator of iron homeostasis relevant in anemia, has recently entered phase-1 clinical trials sponsored by Pieris Pharmaceuticals, Inc. (http://www.pieris.com). Another Anticalin based on the Tlc scaffold, which was developed to effectively block the vascular endothelial growth factor (VEGF-A), was previously investigated as an inhibitor of tumor angiogenesis in a first-inhuman phase-I trial, constituting a drug candidate for the treatment of solid cancers (Angiocal). Notably, this engineered lipocalin did not show any immunogenicity, as judged by the absence of antidrug antibody responses in 24 patients, including one patient who had received altogether 17 doses.76 H


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