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.     

Exon-intron structure and evolution of the Lipocalin gene family

The Lipocalins are an ancient protein family whose expression is currently confirmed in bacteria, protoctists, plants, arthropods, and chordates. The evolution of this protein family has been assessed previously using amino acid sequence phylogenies. In this report we use an independent set of characters derived from the gene structure (exon-intron arrangement) to infer a new lipocalin phylogeny. We also present the novel gene structure of three insect lipocalins. The position and phase of introns are well preserved among lipocalin clades when mapped onto a protein sequence alignment, suggesting the homologous nature of these introns. Because of this homology, we use the intron position and phase of 23 lipocalin genes to reconstruct a phylogeny by maximum parsimony and distance methods. These phylogenies are very similar to the phylogenies derived from protein sequence. This result is confirmed by congruence analysis, and a consensus tree shows the commonalities between the two source trees. Interestingly, the intron arrangement phylogeny shows that metazoan lipocalins have more introns than other eukaryotic lipocalins, and that intron gains have occurred in the C-termini of chordate lipocalins. We also analyze the relationship of intron arrangement and protein tertiary structure, as well as the relationship of lipocalins with members of the proposed structural superfamily of calycins. Our congruence analysis validates the gene structure data as a source of phylogenetic information and helps to further refine our hypothesis on the evolutionary history of lipocalins.     

Polycalin (chlorophyllid A binding protein): a novel, very large fluorescent lipocalin from the midgut of the domestic silkworm Bombyx mori L

We studied a protein from the midgut of the silkworm Bombyx mori characterized by its ability to bind the prosthetic group of chlorophyll, that confers fluorescent properties to this protein. Several techniques, 2D electrophoresis purification, MS-MS and Maldi-TOF peptide sequencing, RT-PCR and nucleotide sequencing were used to obtain the nucleotide sequence and the deduced amino acid sequence. The coding sequence was compared to the gene sequence to define the number and size of introns and exons.The gene spanned 45.5 kb of DNA and consisted of 46 exons. The cDNA encoded a protein of 2721 amino acids.The protein was identified as a lipocalin with novel features. Most lipocalins are proteins with high affinity to small lipophilic molecules, with a molecular size in the 25 kDa range and a well conserved tertiary structure. The apoprotein described here revealed 15 lipocalin like structures, in line. We called this protein a polycalin (pentadecacalin).     

Genome-wide identification and comparative analysis of lipocalin families in Lepidoptera with an emphasis on Bombyx mori

Lipocalins exhibit functional diversity, including roles in retinol transport, invertebrate cryptic coloration, and stress response. However, genome-wide identification and characterization of lipocalin in the insect lineage have not been thoroughly explored. Here, we found that a lineage-specific expansion of the lipocalin genes in Lepidoptera occurred in large part due to tandem duplication events and several lipocalin genes involving insect coloration were expanded more via tandem duplication in butterflies. A comparative analysis of conserved motifs showed both conservation and divergence of lepidopteran lipocalin family protein structures during evolution. We observe dynamic changes in tissue expression preference of paralogs in Bombyx mori, suggesting differential contribution of paralogs to specific organ functions during evolution. Subcellular localization experiments revealed that lipocalins localize to the cytoplasm, nuclear membrane, or nucleus in BmN cells. Moreover, several lipocalin genes exhibited divergent responses to abiotic and biotic stresses, and 1 lipocalin gene was upregulated by 300 fold in B. mori. These results suggest that lipocalins act as signaling components in defense responses by mediating crosstalk between abiotic and biotic stress responses. This study deepens our understanding of the comprehensive characteristics of lipocalins in insects.     

A lipocalin sequence signature modulates cell survival

Lipocalins are β-barrel proteins, which share three conserved motifs in their amino acid sequence. In this study, we identified by a peptide mapping approach, a seven-amino acid sequence related to one of these motifs (motif 2) that modulates cell survival. A synthetic peptide based on an insect lipocalin displayed cytoprotective activity in serum-deprived endothelial cells and leucocytes. This activity was dependent on nitric oxide synthase. This sequence was found within several lipocalins, including apolipoprotein D, retinol binding protein, lipocalin-type prostaglandin D synthase, and many unknown proteins, suggesting that it is a sequence signature and a lipocalin conserved property.     

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.     

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.     

cDNA cloning and sequencing reveals human tear prealbumin to be a member of the lipophilic-ligand carrier protein superfamily.

The gene encoding human tear prealbumin, a major component of the protein fraction of tear fluid, was cloned from total cDNA of lacrimal gland by polymerase chain reaction using synthetic oligonucleotides derived from N-terminal amino acid sequences of the purified protein. Sequence analysis and a computer-assisted homology search revealed this protein to be a member of the lipocalin superfamily, consisting of hydrophobic-ligand carriers. The deduced amino acid sequence of tear prealbumin shares 58% identity with von Ebner's gland protein from rat, which is supposed to be involved in taste reception. In addition, significant homology has also been found with other members of the lipocalins, e.g. with beta-lactoglobulin. The predicted secondary structure of tear prealbumin resembles that of beta-lactoglobulin in the number and positions of nine beta-sheets and one alpha-helix at the C-terminal part of the protein, thus indicating a three-dimensional structure similar to beta-lactoglobulin. Protein sequencing revealed that the observed electrophoretic heterogeneity of tear prealbumin is due to subtle differences at the N terminus of the protein. The function of tear prealbumin as a lipophilic carrier was further supported by the fact that it binds [3H]retinol in vitro. Although this protein was originally described to be tear-specific, a tear prealbumin-specific antiserum also reacted with proteins of human saliva, sweat, and nasal mucus.     

GP2/THP gene family of self-binding, GPI-anchored proteins forms a cluster at chromosome 7F1 region in mouse genome

We investigated the genomic organization of pancreatic zymogen granule membrane-associated protein GP2, a GPI-anchored protein exhibiting self-aggregation at acidic pH, in order to construct a gene-knockout mouse. Cloning and analysis of lambda clones encoding GP2 from 129 Svj mouse genomic DNA libraries showed that the GP2 gene spans about 16.8 kb and includes 11 exons. Identifiable functional domains including a signal sequence, an EGF-like motif, a putative condensing ZP domain, a GPI-anchor attachment site, and a transmembrane sequence for GPI anchoring are encoded in separate exons. Using FISH, the GP2 gene was mapped to mouse chromosome 7F1 near the gene for THP, a GP2 homolog expressed in the cells of thick ascending loop of Henle (TALH) in the kidney. Further analysis of the mouse genome revealed that the THP and GP2 genes are adjacent to one another and are separated by only 3.5 kb in the 7F1 locus. Additionally, the overall structure of the THP gene, 16.2kb with 11 exons, was strikingly similar to that of GP2. This finding suggests that the GP2 and THP genes were generated by gene duplication and evolved separately to acquire regulatory elements leading to tissue-specific expression. Comparative analysis revealed that the 5' flanking region of the THP gene is similar to the first intron of NKCC2, a TALH cell-specific ion-transporter gene. The promoter region of the GP2 gene shares cis-elements found in other pancreas-specific genes. Using this genetic information, a GP2 null mutation was successfully introduced into an ES cell line, and an animal model was established without disruption of THP expression.     

Enforced expression of FGF-7 promotes epithelial hyperplasia whereas a dominant negative FGFR2iiib promotes the emergence of neuroendocrine phenotype in prostate glands of transgenic mice

The minimal rat probasin (PB) promoter was used to target expression of human fibroblast growth factor-7 (FGF-7)/keratinocyte growth factor (KGF) directly to prostatic epithelium of transgenic mice, converting FGF-7 from a paracrine to an autocrine factor. Four independent lines were established that expressed the transgene (PKS) in the prostate. Upon histologic analysis, the prostatic epithelium of PKS mice was found to be hyperplastic. Many of the prostatic ducts were filled with secretory epithelial cells tightly associated with a highly enfolded basement membrane. Distortions of the ductal smooth muscle layer were also observed. Prostates from year-old PKS mice had significantly more abnormal ducts than their wild-type nontransgenic littermates. The minimal rat PB promoter was also used to target a truncated FGFR2iiib receptor to prostatic epithelium to functionally abrogate endogenous FGF-7 signaling. Three lines were established that expressed the transgene (KDNR) in the prostate. Upon dissection it was noted that all four lobes of the prostates of KDNR mice were present but smaller in size. Histologic analysis indicated that the epithelium in many of the prostatic ducts was disorganized and contained numerous rounded cytokeratin-positive cells that were not tightly associated with the basement membrane. The stroma was disorganized and did not form a tight layer of smooth muscle around the epithelial ducts. Surprisingly, abrogation of FGF signaling in KDNR mice correlated with the emergence of a neuroendocrine-like phenotype that was not observed as a consequence of enforced FGF-7 expression in the PKS mice.     

Genomic organization and chromosomal localization of the murine epididymal retinoic acid-binding protein (mE-RABP) gene

The murine epididymal retinoic acid-binding protein (mE-RABP) is specifically synthesized in the mouse mid/distal caput epididymidis and secreted in the lumen. In this report, we have demonstrated by Southern blot analysis of genomic DNA that mE-RABP is encoded by a single-copy gene. A mouse 129/SvJ genomic bacterial artificial chromosome (BAC) library was screened using a cDNA encoding the minor form of mE-RABP. One positive BAC clone was characterized and sequenced to determine the nucleotide sequence of the entire mE-RABP gene. The molecular cloning of the mE-RABP gene completes the characterization of the 20.5-kDa–predicted preprotein leading to the minor and major forms of mE-RABP. Comparison of the DNA sequence of the promoter and coding regions with that of the rat epididymal secretory protein I (ESP I) gene showed that the mE-RABP gene is the orthologue of the ESP I gene that encodes a rat epididymal retinoic acid-binding protein. Several regulatory elements, including a putative androgen receptor binding site, “CACCC-boxes,” NF-1, Oct-1, and SP-1 recognition sites, are conserved in the proximal promoter. Analysis of the nucleotide sequence of the mE-RABP gene revealed the presence of seven exons and showed that the genomic organization is highly related to other genes encoding lipocalins. The mE-RABP gene was mapped by fluorescent in situ hybridization to the [A3-B] region of the murine chromosome 2. Our data, combined with that of others, suggest that the proximal segment of the mouse chromosome 2 may be a rich region for genes encoding lipocalins with a genomic organization highly related to the mE-RABP gene. Mol. Reprod. Dev. 50:387–395, 1998. © 1998 Wiley-Liss, Inc.