Exon-intron structure of outlier tick lipocalins indicate a monophyletic origin within the larger lipocalin family

All tick proteins assigned to the lipocalin family lack the structural conserved regions (SCRs) that are characteristic of the kernel lipocalins and can thus be classified as outliers. These tick proteins have been assigned to the tick lipocalin family based on database searches that indicated homology between tick sequences and the fact that the histamine binding protein (HBP2) from the hard tick Rhipicephalus appendiculatus (Ixodidae) shows structural similarity to the lipocalin fold. Sequence identity between kernel and outlier lipocalins falls below 20% and the question raised is whether the outlier and kernel lipocalins are truly homologous. More specifically in the case of the tick lipocalins, whether their structural fold is derived from the lipocalin fold or whether convergent evolution resulted in the generation of the basic lipocalin-like fold which consists of an eight stranded continuous anti-parallel beta-barrel terminated by a C-terminal alpha-helix that lies parallel to the barrel. The current study determined the gene structure for HBP2 and TSGP1, TSGP2 and TSGP4, lipocalins identified from the soft tick Ornithodoros savignyi (Argasidae). All tick lipocalins have four introns (A-D) with conserved positions and phases within the tick lipocalin sequence alignment. The positions and phase information are also conserved with regard to the rest of the lipocalin family. Phylogenetic analysis using this information shows conclusively that tick lipocalins are evolutionary related to the rest of the lipocalin family. Tick lipocalins are grouped within a monophyletic clade that indicates a monophyletic origin within the tick lineage and also group with the other arthropod lipocalins in a larger clade. Phylogenetic analysis of sequence alignments based on conserved secondary structure of the lipocalin fold support the conclusions from the gene structure trees. These results indicate that exon-intron arrangement can be useful for the inclusion of outlier lipocalins within the larger lipocalin family.     

Lipocalins as a scaffold

The concept of scaffolds that can be equipped with artificial biochemically active sites has gained recent interest in the field of protein design. Members of the lipocalin protein family represent promising model systems in this respect. Especially prototypic lipocalins, such as the retinol-binding protein or the bilin-binding protein (BBP), exhibit a structurally simple one-domain fold with a conformationally well conserved beta-barrel as their central motif. This type of supersecondary structure is made of a cylindrically closed beta-sheet of eight antiparallel strands. At the open end of the barrel the beta-strands are connected by four loops in a pairwise manner so that a pocket for the ligand is formed. In a rational protein design study a metal-binding site was functionally grafted on the solvent-exposed surface of the beta-barrel, whereby the rigid backbone conformation permitted the spatially defined arrangement of three His side chains. In a combinatorial protein design approach, the natural ligand pocket of a lipocalin was reshaped. In this manner variants of the BBP were engineered which exhibit high affinity and remarkable specificity for haptens like fluorescein and digoxigenin. The so-called 'anticalins', i.e. artificial lipocalins recognizing prescribed ligands, could provide an interesting alternative to recombinant antibody fragments. Consequently, the use of lipocalins as a scaffold opens new applications for members of this functionally diverse protein family in biotechnology and medicine.     

NMR solution structure of lipocalin-type prostaglandin D synthase: evidence for partial overlapping of catalytic pocket and retinoic acid-binding pocket within the central cavity

Lipocalin-type prostaglandin (PG) D synthase (L-PGDS) catalyzes the isomerization of PGH(2), a common precursor of various prostanoids, to produce PGD(2), an endogenous somnogen and nociceptive modulator, in the brain. L-PGDS is a member of the lipocalin superfamily and binds lipophilic substances, such as retinoids and bile pigments, suggesting that L-PGDS is a dual functional protein acting as a PGD(2)-synthesizing enzyme and a transporter for lipophilic ligands. In this study we determined by NMR the three-dimensional structure of recombinant mouse L-PGDS with the catalytic residue Cys-65. The structure of L-PGDS exhibited the typical lipocalin fold, consisting of an eight-stranded, antiparallel beta-barrel and a long alpha-helix associated with the outer surface of the barrel. The interior of the barrel formed a hydrophobic cavity opening to the upper end of the barrel, the size of which was larger than those of other lipocalins, and the cavity contained two pockets. Molecular docking studies, based on the result of NMR titration experiments with retinoic acid and PGH(2) analog, revealed that PGH(2) almost fully occupied the hydrophilic pocket 1, in which Cys-65 was located and all-trans-retinoic acid occupied the hydrophobic pocket 2, in which amino acid residues important for retinoid binding in other lipocalins were well conserved. Mutational and kinetic studies provide the direct evidence for the PGH(2) binding mode. These results indicated that the two binding sites for PGH(2) and retinoic acid in the large cavity of L-PGDS were responsible for the broad ligand specificity of L-PGDS and the non-competitive inhibition of L-PGDS activity by retinoic acid.     

Small angle X-ray scattering analysis of ligand-bound forms of tetrameric apolipoprotein-D

Human apolipoprotein-D (apoD) is a glycosylated lipocalin that plays a protective role in Alzheimer’s disease due to its antioxidant function. Native apoD from human body fluids forms oligomers, predominantly a stable tetramer. As a lipocalin, apoD binds and transports small hydrophobic molecules such as progesterone, palmitic acid and sphingomyelin. Oligomerisation is a common trait in the lipocalin family and is affected by ligand binding in other lipocalins. The crystal structure of monomeric apoD shows no major changes upon progesterone binding. Here, we used small-angle X-ray scattering (SAXS) to investigate the influence of ligand binding and oxidation on apoD oligomerisation and conformation. As a solution-based technique, SAXS is well suited to detect changes in oligomeric state and conformation in response to ligand binding. Our results show no change in oligomeric state of apoD and no major conformational changes or subunit rearrangements in response to binding of ligands or protein oxidation. This highlights the highly stable structure of the native apoD tetramer under various physiologically relevant experimental conditions.     

Evolution of the lipocalin family as inferred from a protein sequence phylogeny

The lipocalins constitute a family of proteins that have been found in eubacteria and a variety of eukaryotic cells, where they play diverse physiological roles. It is the primary goal of this review to examine the patterns of change followed by lipocalins through their complex history, in order to stimulate scientists in the field to experimentally contrast our phylogeny-derived hypotheses. We reexamine our previous work on lipocalin phylogeny and update the phylogenetic analysis of the family. Lipocalins separate into 14 monophyletic clades, some of which are grouped in well supported superclades. The lipocalin tree was rooted with the bacterial lipocalin genes under the assumption that they have evolved from a single common ancestor with the metazoan lipocalins, and not by horizontal transfer. The topology of the rooted tree and the species distribution of lipocalins suggest that the newly arising lipocalins show a higher rate of amino acid sequence divergence, a higher rate of gene duplication, and their internal pocket has evolved towards binding smaller hydrophobic ligands with more efficiency.     

A phylogenetic analysis of the lipocalin protein family

The lipocalins are a family of extracellular proteins that bind and transport small hydrophobic molecules. They are found in eubacteria and a great variety of eukaryotic cells, in which they play diverse physiological roles. We report here the detection of two new eukaryotic lipocalins and a phylogenetic analysis of 113 lipocalin family members performed with maximum-likelihood and parsimony methods on their amino acid sequences. Lipocalins segregate into 13 monophyletic clades, some of which are grouped in well-supported superclades. An examination of the G + C content of the bacterial lipocalin genes and the detection of four new conceptual lipocalins in other eubacterial species argue against a recent horizontal transfer as the origin of prokaryotic lipocalins. Therefore, we rooted our lipocalin tree using the clade containing the prokaryotic lipocalins. The topology of the rooted lipocalin tree is in general agreement with the currently accepted view of the organismal phylogeny of arthropods and chordates. The rooted tree allows us to assign polarity to character changes and suggests a plausible scenario for the evolution of important lipocalin properties. More recently evolved lipocalins tend to (1) show greater rates of amino acid substitutions, (2) have more flexible protein structures, (3) bind smaller hydrophobic ligands, and (4) increase the efficiency of their ligand-binding contacts. Finally, we found that the family of fatty-acid-binding proteins originated from the more derived lipocalins and therefore cannot be considered a sister group of the lipocalin family.     

Protein-segment universe exhibiting transitions at intermediate segment length in conformational subspaces

Background

Lipocalins are widely distributed in nature and are found in bacteria, plants, arthropoda and vertebra. In hematophagous arthropods, they are implicated in the successful accomplishment of the blood meal, interfering with platelet aggregation, blood coagulation and inflammation and in the transmission of disease parasites such as Trypanosoma cruzi and Borrelia burgdorferi. The pairwise sequence identity is low among this family, often below 30%, despite a well conserved tertiary structure. Under the 30% identity threshold, alignment methods do not correctly assign and align proteins. The only safe way to assign a sequence to that family is by experimental determination. However, these procedures are long and costly and cannot always be applied. A way to circumvent the experimental approach is sequence and structure analyze. To further help in that task, the residues implicated in the stabilisation of the lipocalin fold were determined. This was done by analyzing the conserved interactions for ten lipocalins having a maximum pairwise identity of 28% and various functions.

Results

It was determined that two hydrophobic clusters of residues are conserved by analysing the ten lipocalin structures and sequences. One cluster is internal to the barrel, involving all strands and the 3₁₀ helix. The other is external, involving four strands and the helix lying parallel to the barrel surface. These clusters are also present in RaHBP2, a unusual "outlier" lipocalin from tick Rhipicephalus appendiculatus. This information was used to assess assignment of LIR2 a protein from Ixodes ricinus and to build a 3D model that helps to predict function. FTIR data support the lipocalin fold for this protein.

Conclusion

By sequence and structural analyzes, two conserved clusters of hydrophobic residues in interactions have been identified in lipocalins. Since the residues implicated are not conserved for function, they should provide the minimal subset necessary to confer the lipocalin fold. This information has been used to assign LIR2 to lipocalins and to investigate its structure/function relationship. This study could be applied to other protein families with low pairwise similarity, such as the structurally related fatty acid binding proteins or avidins.     

Structure and sequence relationships in the lipocalins and related proteins.

The lipocalins and fatty acid-binding proteins (FABPs) are two recently identified protein families that both function by binding small hydrophobic molecules. We have sought to clarify relationships within and between these two groups through an analysis of both structure and sequence. Within a similar overall folding pattern, we find large parts of the lipocalin and FABP structures to be quantitatively equivalent. The three largest structurally conserved regions within the lipocalin common core correspond to characteristic sequence motifs that we have used to determine the constitution of this family using an iterative sequence analysis procedure. This afforded a new interpretation of the family, which highlighted the difficulties of determining a comprehensive and coherent classification of the lipocalins. The first of the three conserved sequence motifs is also common to the FABPs and corresponds to a conserved structural element characteristic of both families. Similarities of structure and sequence within the two families suggests that they form part of a larger "structural superfamily"; we have christened this overall group the calycins to reflect the cup-shaped structure of its members.     

Crystal structure of the allergen Equ c 1. A dimeric lipocalin with restricted IgE-reactive epitopes

The three-dimensional structure of the major horse allergen Equ c 1 has been determined at 2.3 A resolution by x-ray crystallography. Equ c 1 displays the typical fold of lipocalins, a beta-barrel flanked by a C-terminal alpha-helix. The space between the two beta-sheets of the barrel defines an internal cavity that could serve, as in other lipocalins, for the binding and transport of small hydrophobic ligands. Equ c 1 crystallizes in a novel dimeric form, which is distinct from that observed in other lipocalin dimers and corresponds to the functional form of the allergen. Binding studies of point mutants of the allergen with specific monoclonal antibodies raised in mouse and IgE serum from horse allergic patients allowed to identify putative B cell antigenic determinants. In addition, total inhibition of IgE serum recognition by a single specific monoclonal antibody revealed the restricted nature of the IgE binding target on the molecular surface of Equ c 1.     

<Emphasis Type="Italic">In Silico</Emphasis> Study on Retinoid-binding Modes in Human RBP and ApoD Lipocalins

Lipocalins are proteins with highly homologous structures but diverse sequences that are potential candidates for scaffold protein engineering with novel ligand-binding functions. Numerous crystal structures of lipocalin-ligand complexes have been identified and used in the study of their binding modes. On the other hand, crystallization studies cannot meet the increasing demand for novel lipocalin-ligand complexes in scaffold engineering, which requires rapid computational analyses of their binding modes in parallel. Human retinol-binding protein (RBP) and apolipoprotein D (apoD) are sequentially very distant proteins, but they show tight binding against retinoids, such as retinol and retinoic acid. In the present study, complexes of the two lipocalins with retinol and retinoic acid were modeled computationally by a molecular docking simulation, and their ligand-binding modes were analyzed at a molecular level. The models identified the crucial residues of lipocalins that interact with the ligands and revealed the similarities and differences in their retinoid-binding modes as well as in the specific interactions of the retinoid species within the same lipocalin. An analysis of the amino acid propensity of the retinoid-binding residues suggested that the evolutionary preference of the residues is restricted to the binding pocket rather than the entire protein. The distribution of charged residues around the terminus of retinoic acid showed a huge difference between RBP and ApoD, which might be a factor for the different binding affinities of lipocalins against retinoic acid. This in silico study is expected to be applied to scaffold protein engineering for novel retinoid-binding lipocalins.     

Similarity of Binding Potentials Between Plant DUF538 and Animal Lipocalin: Cholesterol Binding Ability of DUF538

Objective DUF538(domain of unknown function 538) domain containing proteins are known as putative hypothetical proteins in plants. Until yet, there is no much information regarding their structure and function. Methods In the present research work, the homologous structures and binding potentials were identified between plant/mammalian lipocalins and plant DUF538 protein by using bioinformatics and experimental tools including molecular dynamics simulation, molecular docking and recombinant tech...     

Comparative ligand-binding analysis of ten human lipocalins

At least ten different lipocalins occur in the human body: retinol-binding protein (RBP), alpha1-acid glycoprotein, alpha1-microglobulin, apolipoprotein D, beta-trace protein, complement component 8gamma, glycodelin, neutrophil gelatinase-associated lipocalin, odorant-binding protein, and tear lipocalin. Although many of these lipocalins seem to play an important physiological role, their precise biological function is not always clear. Especially the interpretation of their diverse ligand-binding activities has been hampered by the fact that the natural lipocalins were prepared from different sources and with varying purity. Here we present a generic expression and purification strategy for the recombinant lipocalins, which is based on secretion into the periplasm of E. coli, where disulphide bonds are readily formed, followed by affinity purification via the Strep-tag II and gel filtration. The ten human lipocalins were successfully prepared and their ligand-binding activities were compared via fluorescence titration with a set of typical ligands: retinol, retinoic acid (RA), 11-(5-(dimethylamino)-1-naphthalene-sulfonylamino)undecanoic acid (DAUDA), and 8-anilino-1-naphtalene-sulfonic acid (ANS). As result, merely two lipocalins, RBP and beta-trace, revealed high affinities both for retinol and for RA, which probably reflects a specialized physiological function in retinoid complexation. Surprisingly, the strongest retinol affinity was detected for apolipoprotein D, whereas this lipocalin exhibits much weaker binding activity for retinoic acid. Binding studies with the two spectroscopic probes DAUDA and ANS revealed mixed patterns, which demonstrates that the affinity for lipophilic substances varies considerably among human lipocalins. Notably, RBP with its perfectly moulded retinol-binding site did not show any detectable binding activity for both compounds. Hence, our recombinant expression and purification system should be useful for further structural and functional studies of lipocalins from human origin and beyond.     

Structure-Based Phylogenetic Analysis of the Lipocalin Superfamily

Lipocalins constitute a superfamily of extracellular proteins that are found in all three kingdoms of life. Although very divergent in their sequences and functions, they show remarkable similarity in 3-D structures. Lipocalins bind and transport small hydrophobic molecules. Earlier sequence-based phylogenetic studies of lipocalins highlighted that they have a long evolutionary history. However the molecular and structural basis of their functional diversity is not completely understood. The main objective of the present study is to understand functional diversity of the lipocalins using a structure-based phylogenetic approach. The present study with 39 protein domains from the lipocalin superfamily suggests that the clusters of lipocalins obtained by structure-based phylogeny correspond well with the functional diversity. The detailed analysis on each of the clusters and sub-clusters reveals that the 39 lipocalin domains cluster based on their mode of ligand binding though the clustering was performed on the basis of gross domain structure. The outliers in the phylogenetic tree are often from single member families. Also structure-based phylogenetic approach has provided pointers to assign putative function for the domains of unknown function in lipocalin family. The approach employed in the present study can be used in the future for the functional identification of new lipocalin proteins and may be extended to other protein families where members show poor sequence similarity but high structural similarity.     

The solution structure and dynamics of human neutrophil gelatinase-associated lipocalin.

Human neutrophil gelatinase-associated lipocalin (HNGAL) is a member of the lipocalin family of extracellular proteins that function as transporters of small, hydrophobic molecules. HNGAL, a component of human blood granulocytes, binds bacterially derived formyl peptides that act as chemotactic agents and induce leukocyte granule discharge. HNGAL also forms a complex with the proenzyme form of matrix metalloproteinase-9 (pro-MMP-9, or progelatinase B) via an intermolecular disulphide bridge. This association allows the subsequent formation of ternary and quaternary metalloproteinase/inhibitor complexes that vary greatly in their metalloproteinase activities. The structure and dynamics of apo-HNGAL have been determined by NMR spectroscopy. Simulated annealing calculations yielded a set of 20 convergent structures with an average backbone RMSD from mean coordinate positions of 0. 79(+/-0.13) A over secondary structure elements. The overall rotational correlation time (13.3 ns) derived from15N relaxation data is consistent with a monomeric protein of the size of HNGAL (179 residues) under the experimental conditions (1.4 mM protein, pH 6.0, 24.5 degrees C). The structure features an eight-stranded antiparallel beta-barrel, typical of the lipocalin family. One end of the barrel is open, providing access to the binding site within the barrel cavity, while the other is closed by a short 310-helix. The free cysteine residue required for association with pro-MMP-9 lies in an inter-strand loop at the closed end of the barrel. The structure provides a detailed model of the ligand-binding site and has led to the proposal of a site for pro-MMP-9 association. Dynamic data correlate well with structural features, which has allowed us to investigate a mechanism by which a cell-surface receptor might distinguish between apo and holo-HNGAL through conformational changes at the open end of the barrel.     

'Anticalins': a new class of engineered ligand-binding proteins with antibody-like properties

The development of soluble receptor proteins that recognise given target molecules--ranging from small chemical compounds to macromolecular structures at a cell surface, for example--is of ever increasing importance in the life sciences and biotechnology. For the past century this area of application was dominated by antibodies, which were traditionally generated via immunisation of animals but have recently also become available by means of protein engineering methods. The so-called 'anticalins' offer an alternative type of ligand-binding proteins, which has been constructed on the basis of lipocalins as a scaffold. The central element of this protein architecture is a beta-barrel structure of eight antiparallel strands, which supports four loops at its open end. These loops form the natural binding site of the lipocalins and can be reshaped in vitro by extensive amino acid replacement, thus creating novel binding specificities. The bilin-binding protein (BBP) was employed as a model system for the preparation of a random library with 16 selectively mutagenized residues. Using bacterial phagemid display and colony screening techniques, several lipocalin variants--termed anticalins--have been selected from this library, exhibiting binding activity for compounds like fluorescein or digoxigenin. Anticalins possess high affinity and specificity for their prescribed ligands as well as fast binding kinetics, so that their functional properties are similar to those of antibodies. Compared with them, they exhibit however several advantages, including a smaller size, composition of a single polypeptide chain, and a simple set of four hypervariable loops that can be easily manipulated at the genetic level. Apart from haptenic compounds as targets, anticalins should also be able to recognise macromolecular antigens, provided that the random library is accordingly designed. Hence, they should not only serve as valuable reagents for bioanalytical purposes, but may also have a potential in replacing antibodies for medical therapy.     

‘Anticalins’: a new class of engineered ligand-binding proteins with antibody-like properties

The development of soluble receptor proteins that recognise given target molecules — ranging from small chemical compounds to macromolecular structures at a cell surface, for example — is of ever increasing importance in the life sciences and biotechnology. For the past century this area of application was dominated by antibodies, which were traditionally generated via immunisation of animals but have recently also become available by means of protein engineering methods. The so-called ‘anticalins’ offer an alternative type of ligand-binding proteins, which has been constructed on the basis of lipocalins as a scaffold. The central element of this protein architecture is a β-barrel structure of eight antiparallel strands, which supports four loops at its open end. These loops form the natural binding site of the lipocalins and can be reshaped in vitro by extensive amino acid replacement, thus creating novel binding specificities. The bilin-binding protein (BBP) was employed as a model system for the preparation of a random library with 16 selectively mutagenized residues. Using bacterial phagemid display and colony screening techniques, several lipocalin variants — termed anticalins — have been selected from this library, exhibiting binding activity for compounds like fluorescein or digoxigenin. Anticalins possess high affinity and specificity for their prescribed ligands as well as fast binding kinetics, so that their functional properties are similar to those of antibodies. Compared with them, they exhibit however several advantages, including a smaller size, composition of a single polypeptide chain, and a simple set of four hypervariable loops that can be easily manipulated at the genetic level. Apart from haptenic compounds as targets, anticalins should also be able to recognise macromolecular antigens, provided that the random library is accordingly designed. Hence, they should not only serve as valuable reagents for bioanalytical purposes, but may also have a potential in replacing antibodies for medical therapy.     

Crystal structure of the human odorant binding protein,OBPIIa

Human odorant‐binding protein, OBP_IIa, is expressed by nasal epithelia to facilitate transport of hydrophobic odorant molecules across the aqueous mucus. Here, we report its crystallographic analysis at 2.6 Å resolution. OBP_IIa is a monomeric protein that exhibits the classical lipocalin fold with a conserved eight‐stranded β‐barrel harboring a remarkably large hydrophobic pocket. Basic residues within the four loops that shape the entrance to this ligand‐binding site evoke a positive electrostatic potential. Human OBP_IIa shows distinct features compared with other mammalian OBPs, including a potentially reactive Cys side chain within its pocket similar to human tear lipocalin. Proteins 2015; 83:1180–1184. © 2015 Wiley Periodicals, Inc.     

The crystal structure of human α(1)-microglobulin reveals a potential haem-binding site

We describe the 2.3 ? (1 ?=0.1?nm) X-ray structure of α1m (α1-microglobulin), an abundant protein in human blood plasma, which reveals the β-barrel fold typical for lipocalins with a deep pocket lined by four loops at its open rim. Loop #1 harbours the residue Cys34 which is responsible for covalent cross-linking with plasma IgA. A single disulfide bond between Cys72 and Cys169 connects the C-terminal segment to the β-barrel, as in many other lipocalins. The exposed imidazole side chains of His122 and His123 in loop #4 give rise to a double Ni2+-binding site together with a crystallographic neighbour. The closest structural relatives of α1m are the complement protein component C8γ, the L-prostaglandin D synthase and lipocalin 15, three other structurally characterized members of the lipocalin family in humans that have only distant sequence similarity. In contrast with these, α1m is initially expressed as a bifunctional fusion protein with the protease inhibitor bikunin. Neither the electron density nor ESI-MS (electrospray ionization MS) provide evidence for a chromophore bound to the recombinant α1m, also known as 'yellow/brown lipocalin'. However, the three side chains of Lys92, Lys118 and Lys130 that were reported to be involved in covalent chromophore binding appear to be freely accessible to ligands accommodated in the hydrophobic pocket. A structural feature similar to the well-known Cys-Pro haem-binding motif indicates the presence of a haem-binding site within the loop region of α1m, which explains previous biochemical findings and supports a physiological role in haem scavenging, as well as redox-mediated detoxification.     

Savicalin,a lipocalin from hemocytes of the soft tick, <Emphasis Type="Italic">Ornithodoros savignyi</Emphasis>

Savicalin, is a lipocalin found in the hemocytes of the soft tick, Ornithodoros savignyi. It could be assigned to the tick lipocalin family based on BLAST analysis. Savicalin is the first non-salivary gland lipocalin described in ticks. The mature sequence is composed of 188 amino acids with a molecular mass of 21481.9 Da. A homolog for savicalin was found in a whole body EST-library from a related soft tick O. porcinus, while other tick salivary gland derived lipocalins retrieved from the non-redundant sequence database are more distantly related. Homology modeling supports the inclusion of savicalin into the lipocalin family. The model as well as multiple alignments suggests the presence of five disulphide bonds. Two conserved disulphide bonds are found in hard and soft tick lipocalins. A third disulphide bond is shared with the TSGP4-clade of leukotriene C4 binding soft tick lipocalins and a fourth is shared with a lipocalin from the hard tick Ixodes scapularis. The fifth disulphide bond is unique and links strands D-E. Phylogenetic analysis showed that savicalin is a distant relative of salivary gland derived lipocalins, but groups within a clade that is possibly non-salivary gland derived. It lacks the biogenic amine-binding motif associated with tick histamine and serotonin binding proteins. Expression profiles indicate that savicalin is found in hemocytes, midgut and ovaries, but not in the salivary glands. Up-regulation occurs in hemocytes after bacterial challenge and in midguts and ovaries after feeding. Given its tissue distribution and up-regulation of expression, it is possible that this lipocalin functions in tick development after feeding or in an anti-microbial capacity.     

The 1.8-A crystal structure of human tear lipocalin reveals an extended branched cavity with capacity for multiple ligands

In contrast with earlier assumptions, which classified human tear lipocalin (Tlc) as an outlier member of the lipocalin protein family, the 1.8-A resolution crystal structure of the recombinant apoprotein confirms the typical eight-stranded antiparallel beta-barrel architecture with an alpha-helix attached to it. The fold of Tlc most closely resembles the bovine dander allergen Bos d 2, a well characterized prototypic lipocalin, but also reveals similarity with beta-lactoglobulin. However, compared with other lipocalin structures Tlc exhibits an extremely wide ligand pocket, whose entrance is formed by four partially disordered loops. The cavity deeply extends into the beta-barrel structure, where it ends in two distinct lobes. This unusual structural feature explains the known promiscuity of Tlc for various ligands, with chemical structures ranging from lipids and retinoids to the macrocyclic antibiotic rifampin and even to microbial siderophores. Notably, earlier findings of biological activity as a thiol protease inhibitor have no correspondence in the three-dimensional structure of Tlc, rather it appears that its proteolytic fragments could be responsible for this phenomenon. Hence, the present structural analysis sheds new light on the ligand binding activity of this functionally obscure but abundant human lipocalin.