PLUNC (palate, lung and nasal epithelium clone) proteins make up the largest branch of the BPI (bactericidal/permeability-increasing protein)/LBP (lipopolysaccharide-binding protein) family of lipid-transfer proteins. PLUNCs make up one of the most rapidly evolving mammalian protein families and exhibit low levels of sequence similarity coupled with multiple examples of species-specific gene acquisition and gene loss. Vertebrate genomes contain multiple examples of genes that do not meet our original definition of what is required to be a member of the PLUNC family, namely conservation of exon numbers/sizes, overall protein size, genomic location and the presence of a conserved disulfide bond. This suggests that evolutionary forces have continued to act on the structure of this conserved domain in what are likely to be functionally important ways.

Introduction

PLUNC (palate, lung and nasal epithelium clone) was first described in the nasal epithelium of the mouse embryo and the trachea/bronchi of adult mice [1]. During studies characterizing human and mouse PLUNC, we made the serendipitous discovery of a family of related genes present in a single locus on chromosome 20 [24]. At that time, owing to the less than complete assembly of this region of chromosome 20, along with the smaller size of the EST (expressed sequence tag) database, we were able to predict the existence of seven genes in this locus [3]. We achieved this using the following observations. (i) All of the predicted proteins were shown to exhibit both sequence and structural similarity to members of a lipid-transfer protein family made up of BPI (bactericidal/permeability-increasing protein), LBP (lipopolysaccharide-binding protein), PLTP (phospholipid-transfer protein) and CETP (cholesteryl ester-transfer protein) [4]. (ii) All of the new proteins showed a high degree of conservation of genomic organization and of exon sizes. (iii) All of the proteins conserved the two cysteine residues found in other related proteins. In fact, a key observation was that the second conserved cysteine residue was encoded by nucleotides 4–6 in exon 7 [3,4]. As genomic and EST resources improved and by performing comparative analysis, we were able to more fully expand the locus to identify more human genes and to expand our studies into other species, including birds [57]. It is now possible to identify 11 genes in the human locus and 14 in the locus in mice, with the most recently identified addition to the family being rodent Vom (vomeromodulin)/ryf3 [8,9]. We have previously described in detail how the PLUNC branch of the wider protein family is the largest and is clearly distinct from the remaining members [5,7]. It is the study of this branch of the family that we are most interested in. We have revisited the PLUNC gene loci to generate what we believe to be the complete set of PLUNC genes in human and mouse. The updated loci from these species are presented in Figure 1 and the exonic organization is shown in Table 1, which illustrates the highly divergent nature of the gene family and shows how distinct genes have evolved in distinct lineages. Both loci contain lineage-restricted paralogues and pseudogenes, evidence of the evolutionary activity that has made them one of the most rapidly evolving mammalian genes [4,5]. A close analysis of these loci and other PLUNC family members from different species suggests that it is the SPLUNC portion of the family that is the most diverse [5,6]. These observations are described in greater detail in other papers in this issue of Biochemical Society Transactions [10,11].

Organization of the human and mouse BPIF gene loci

Figure 1
Organization of the human and mouse BPIF gene loci

The upper panel represents the human locus, whereas the lower panel represents the mouse locus. Arrows between the two loci indicate orthologous relationships between individual genes. The position of the locus on the chromosome (Chr) is indicated by the arrowheads and nucleotide numbers on human chromosome 20 [February 2009 human reference sequence (GRCh37) was produced by the Genome Reference Consortium] and mouse chromosome 2 [July 2007 mouse (Mus musculus) genome (NCBI37/mm9)] assembly by NCBI (National Center for Biotechnology Information) and the Mouse Genome Sequencing Consortium, obtained from the UCSC BLAT genome browser (http://genome.ucsc.edu/). BPIFA (SPLUNC) genes are indicated by grey boxes, while BPIFB (LPLUNC) genes are indicated by white boxes. Mouse Bpifa6 (mSPLUNC6) is represented by a graduated box indicating the size of its intermediates. Pseudogenes (P or -ps) are indicated by shaded boxes. Both loci are flanked by the unrelated genes SPA4L and CDK5RAP1 (green). Scale bar, 200 kb.

Figure 1
Organization of the human and mouse BPIF gene loci

The upper panel represents the human locus, whereas the lower panel represents the mouse locus. Arrows between the two loci indicate orthologous relationships between individual genes. The position of the locus on the chromosome (Chr) is indicated by the arrowheads and nucleotide numbers on human chromosome 20 [February 2009 human reference sequence (GRCh37) was produced by the Genome Reference Consortium] and mouse chromosome 2 [July 2007 mouse (Mus musculus) genome (NCBI37/mm9)] assembly by NCBI (National Center for Biotechnology Information) and the Mouse Genome Sequencing Consortium, obtained from the UCSC BLAT genome browser (http://genome.ucsc.edu/). BPIFA (SPLUNC) genes are indicated by grey boxes, while BPIFB (LPLUNC) genes are indicated by white boxes. Mouse Bpifa6 (mSPLUNC6) is represented by a graduated box indicating the size of its intermediates. Pseudogenes (P or -ps) are indicated by shaded boxes. Both loci are flanked by the unrelated genes SPA4L and CDK5RAP1 (green). Scale bar, 200 kb.

Table 1
Conservation of exon sizes between members of the wider BPIF gene family

The sizes of individual exons (in bp) for the human and mouse BPIF genes are compared with those of human BPI, LBP, PLTP, BPIFC (BPIL2) and CETP. Exon sizes were taken from Ensembl files (http://www.ensembl.org/index.html) with some manual determinations of 5′ exons being made from alignments of ESTs on the UCSC BLAT genome browsers (http://genome.ucsc.edu/). Genes that significantly diverge from the others and that are featured in the text are highlighted in boldface. In most of the genes, the translation start site is found in exon 2, with the exceptions being Bpifa6 (mSPLUNC6) and BPIFB4 (LPLUNC4). The stop codon is always found in the final exon. Exon numbering follows the convention outlined previously [4].

Gene Exon… 2 10 11 12 13 14 15 16 
BPIFA1 (SPLUNC1)  56 175 160 108 153 85 64 75 163        
Bpifa1 (mSPLUNC1 46 246 160 108 153 79 64 75 171        
BPIFA2 (SPLUNC2)  55 168 145 108 153 82 64 78 188        
Bpifa2e (mPSP 47 129 142 108 153 82 64 75 172        
BPIFA3 (SPLUNC3)   337 151 108 150 85 64 221         
Bpifa3 (mSPLUNC3  316 151 108 150 85 64 217         
BPIFA4 (BASE)  37 114 148 105 150 81 64 80 416        
Bpifa5 (mSPLUNC5 51 226 160 108 153 79 64 75 160        
Bpifa6 (mSPLUNC6) 39 100 212 287 105 141 64 61 92 43 428       
BPIFB1 (LPLUNC1)  120 156 142 108 150 82 64 86 180 54 159 68 46 64 77 170 
Bpifb1 (mLPLUNC1 40 157 139 108 150 82 64 86 180 54 159 68 46 64 77 163 
BPIFB2 (LPLUNC2)  160 143 94 105 147 61 61 92 186 54 171 68 46 64 77 427 
Bpifb2 (mLPLUNC2 75 144 94 105 147 61 61 92 189 54 171 68 46 64 77 280 
BPIFB3 (LPLUNC3)   217 157 105 141 64 61 92 180 54 171 68 43 64 77 53 
Bpifb3 (mLPLUNC3 126 129 157 105 141 64 61 92 180 54 171 68 43 64 77 289 
BPIFB4 (LPLUNC4)  121 63 508 105 144 64 61 92 201 54 171 68 43 64 77 388 
Bpifb4 (mLPLUNC456 121 63 514 105 144 64 61 92 204 54 171 68 43 64 77 519 
Bpifb5 (mLPLUNC5  152 217 105 132 85 64 86 183 54 150 68 46 64 80 151 
BPIFB6 (LPLUNC6)   97 100 105 150 64 61 92 177 54 174 68 46 64 77 30 
Bpifb6 (mLPLUNC632 106 108 100 105 150 55 61 92 174 51 174 68 46 64 77 206 
Bpifb9 (Vom)  61 280 505 102 156 85 58 71 147  123 68 31 61 80 237 
BPI   231 115 129 162 64 64 92 177 60 168 68 43 64 77 360 
LBP   285 115 129 156 64 64 92 177 60 168 68 43 64 77 399 
PLTP  69 111 100 129 156 64 64 92 177 60 165 68 43 64 77 304 
CETP   248 115 135 71+88 70 61 92 180 51 165 68 34 73 86 254 
BPIFC (BPIL2)  111 124 121 129 156 64 61 92 177 54 171 68 43 64 77 579 
Gene Exon… 2 10 11 12 13 14 15 16 
BPIFA1 (SPLUNC1)  56 175 160 108 153 85 64 75 163        
Bpifa1 (mSPLUNC1 46 246 160 108 153 79 64 75 171        
BPIFA2 (SPLUNC2)  55 168 145 108 153 82 64 78 188        
Bpifa2e (mPSP 47 129 142 108 153 82 64 75 172        
BPIFA3 (SPLUNC3)   337 151 108 150 85 64 221         
Bpifa3 (mSPLUNC3  316 151 108 150 85 64 217         
BPIFA4 (BASE)  37 114 148 105 150 81 64 80 416        
Bpifa5 (mSPLUNC5 51 226 160 108 153 79 64 75 160        
Bpifa6 (mSPLUNC6) 39 100 212 287 105 141 64 61 92 43 428       
BPIFB1 (LPLUNC1)  120 156 142 108 150 82 64 86 180 54 159 68 46 64 77 170 
Bpifb1 (mLPLUNC1 40 157 139 108 150 82 64 86 180 54 159 68 46 64 77 163 
BPIFB2 (LPLUNC2)  160 143 94 105 147 61 61 92 186 54 171 68 46 64 77 427 
Bpifb2 (mLPLUNC2 75 144 94 105 147 61 61 92 189 54 171 68 46 64 77 280 
BPIFB3 (LPLUNC3)   217 157 105 141 64 61 92 180 54 171 68 43 64 77 53 
Bpifb3 (mLPLUNC3 126 129 157 105 141 64 61 92 180 54 171 68 43 64 77 289 
BPIFB4 (LPLUNC4)  121 63 508 105 144 64 61 92 201 54 171 68 43 64 77 388 
Bpifb4 (mLPLUNC456 121 63 514 105 144 64 61 92 204 54 171 68 43 64 77 519 
Bpifb5 (mLPLUNC5  152 217 105 132 85 64 86 183 54 150 68 46 64 80 151 
BPIFB6 (LPLUNC6)   97 100 105 150 64 61 92 177 54 174 68 46 64 77 30 
Bpifb6 (mLPLUNC632 106 108 100 105 150 55 61 92 174 51 174 68 46 64 77 206 
Bpifb9 (Vom)  61 280 505 102 156 85 58 71 147  123 68 31 61 80 237 
BPI   231 115 129 162 64 64 92 177 60 168 68 43 64 77 360 
LBP   285 115 129 156 64 64 92 177 60 168 68 43 64 77 399 
PLTP  69 111 100 129 156 64 64 92 177 60 165 68 43 64 77 304 
CETP   248 115 135 71+88 70 61 92 180 51 165 68 34 73 86 254 
BPIFC (BPIL2)  111 124 121 129 156 64 61 92 177 54 171 68 43 64 77 579 

Although no structure of any PLUNC has been determined, evidence from modelling studies shows that PLUNC proteins subdivide into what we originally described as short (SPLUNC) and long (LPLUNC) proteins [4]. SPLUNCs contain domains structurally similar to the N-terminal domain of BPI, whereas LPLUNCs contain domains structurally similar to both domains of BPI [4]. The two domains of BPI exhibit distinct cellular functions and yet share strong structural similarity while exhibiting very low levels of sequence identity. The functionally important disulfide bond present within the N-terminal domain of BPI [12] was found to be present in all of the PLUNC proteins identified originally and has therefore been assumed to be of functional importance. However, it is important to note that this suggestion remains to be formally tested.

As further members of the wider family have been identified in a range of species [57], it has become increasingly clear that some of the characteristics that we originally used for identification of individual family members are no longer always to be found in all genes. In the present review, we focus on some of the differences that we have identified in a number of these proteins. In doing so, we also highlight how these genes are described in terms of our updated phylogeny whereby all family members are renamed using the root BPIF for ‘BPI fold-containing’. As set out in detail elsewhere [10], in this systematic nomenclature, single-domain-containing family members have the designation BPIFA and two-domain-containing family members have the designation BPIFB.

PLUNC/BPIF genes that exhibit some divergence in either exon size conservation or exon number are illustrated in Table 1. We have previously described in detail how BPIFB4 (LPLUNC4) is an outlier in this gene family [5,13] and in the present review, we discuss in more detail two additional members, BPIFA4 [BASE (breast cancer and salivary gland expression)] and BPIFB9 (Vom), which both stray from our guiding rules. We have focused on these two genes as they have both been independently studied by a number of groups [8,9,14,15,16]. Table 1 also clearly shows that two other genes, BPIFA3 (SPLUNC3) and Bpifa6 (Splunc6), diverge significantly from our rules, but at this time these two have not been studied at all. BPIFA3 appears to be highly restricted to the testes, is represented by multiple EST from a range of species and appears to undergo complex alternative splicing, whereas Bpifa6, which contains 11 exons and may be a further example of a lineage (rodent)-specific family member, is supported by five ESTs derived from mouse eye RNA.

BPIFA4 (BASE)

BPIFA4 was identified as a putative secreted protein that might be used in the diagnosis of breast cancer [14]. BPIFA4 expression was initially found in breast cancer cell lines and tissue, whereas in normal tissue, it was only seen in salivary gland [7]. We showed that BPIFA4 is a SPLUNC and that its closest paralogue at the time was horse latherin (GenBank® accession number AF491288) which is 42% identical [5,15]. The gene appears to be absent from the rodent lineage, but has been identified in cows and cats [5,6,10,16]. The predicted protein product of human BPIFA4 is significantly shorter than other single-domain-containing proteins and appears to be missing approximately 50 amino acids from the C-terminal end of the molecule, including the cysteine residue that is conserved in all other family members [4,5]. Comparative alignment of the genomic sequence from chimpanzee, gorilla and rhesus monkey shows that exon 6 of human BPIFA4 is 1 nt shorter than that from the other three species, suggesting that this loss has occurred since the divergence of humans and chimpanzee from their common ancestor. The consequence of this single nucleotide difference in the human genome is a frameshift that leads to the introduction of the ‘premature’ stop codon. The resultant protein does not encode the second conserved cysteine residue that by analogy to the structure of BPI is predicted to be crucial to the stability of the protein structure [4,5,17]. The predicted structure is also lacking the second α-helix region that makes up part of the hydrophobic cleft seen in other family members (Figure 2). The converse of this is that the primate BPIFA4 genes would encode bona fide single-domain-containing proteins. This was confirmed by analysis of rhesus monkey BPIFA4 cDNAs cloned from salivary gland RNA (GenBank® accession number AY913830) as well as predictions of genomic DNAs of other primates. Consequently, human BPIFA4 appears to be an example of a pseudogene (and should be properly identified as BPIFA4P) that is perhaps better described as a ‘dying’ gene, since it appears to be both transcribed and translated, but no longer encodes a functional protein product. The generation of such pseudogenes specific to the human lineage that contain a single missense mutation in the human orthologue is an exceptionally rare event [18].

Human BPIFA4P (BASE) lacks key structural elements that are found in other PLUNCs

Figure 2
Human BPIFA4P (BASE) lacks key structural elements that are found in other PLUNCs

A modelled structure of chimpanzee BPIFA4 was generated to show the position of the regions predicted to be frame-shifted (cyan) and deleted (red) in the human orthologue. The highly conserved disulfide bond is shown in yellow.

Figure 2
Human BPIFA4P (BASE) lacks key structural elements that are found in other PLUNCs

A modelled structure of chimpanzee BPIFA4 was generated to show the position of the regions predicted to be frame-shifted (cyan) and deleted (red) in the human orthologue. The highly conserved disulfide bond is shown in yellow.

The frameshift in the predicted human BPIFA4 protein removes a long α-helical segment that is predicted to be core to the entire protein fold. Our previous suggestion that the human protein would not fold correctly is supported by our efforts to generate cell lines that secrete the protein. Both human and rhesus monkey BPIFA4 could be detected in the cell lysates from cells stably transfected with epitope-tagged BPIFA4-expressing constructs; however, the medium from a human BPIFA4-expressing cell line did not secrete protein, whereas the medium from the rhesus monkey BASE-expressing cell line did. We have been unable to detect BPIFA4 in primary breast cancer samples by immunohistochemistry (L. Bingle and C.D. Bingle, unpublished work), even though approximately 50% of tumours re-express the gene [14,19].

BPIFB9 (Vom/ryf3)

Vom was originally identified in the lateral nasal glands of rats [8]. The same gene, known as ryf3, was cloned in a study designed to identify genes expressed in the olfactory regions of the rat [9]. Vom is found in the maxillary sinus component of the lateral nasal gland and the vomeronasal organ in rats [20]. The protein is a heavily glycosylated molecule, approximately 70 kDa in size. Owing to the sites at which this protein is found, it has been suggested to have a role in olfaction, although, as with other PLUNC proteins, no compelling functional data have been shown. In our original description of the human BPIF locus, we noted that Vom/ryf3 was located at the telomeric end of the locus [4], but did not pursue the gene any further as there was no human EST support at that time. Subsequently, it was reported that rodent Vom exhibited weak sequence similarity with BPIF proteins [21], and consequently it has emerged that the protein is the most divergent member of the BPIF family (and is named BPIFB9 [10]). Recently, the protein has been shown to be an auto-antigen associated with autoimmune lung disease in aire-deficient mice [22]. Uncovering the relationship of BPIFB9 to other family members was somewhat taxing, as the protein lacks many of the key identification features outlined above. At 591 amino acids, rodent BPIFB9 is much bigger than any of the BPIFB proteins, with the exception of BPIFB4. The extra coding sequence required to create this size of protein means that the exon sizes also do not follow the BPIF pattern (Table 1). Most interestingly, the second cysteine residue, responsible for the formation of the presumed important disulfide bond [4,5,12], is also absent. This is the only case in which a fully functional and secreted member of this protein family is lacking this structural feature. The BPIFB9-coding region is located after BPIFB1 (Figure 1). A comparative analysis of this genomic region shows that BPIFB9 is not present in all mammals and indeed the gene appears to be a pseudogene in a range of species, including humans [18,23], consistent with the lack of human EST data. In addition, the mouse genome contains two highly similar genes, Bpifb9a (4833413D08Rik) and Bpifb9b (5430413K10Rik) that are supported by a significant number of ESTs. The rat genome contains a single gene. This suggests that the two mouse genes are the result of a very recent species-specific duplication that leaves the two proteins with a 98.6% sequence identity. Both isoforms have been identified as major components of mouse lateral nasal glands in proteomic studies [24].

By systematic analysis of the ever-increasing amount of genomic sequence information, we have been able to provide what we now believe is a complete set of BPIF genes in humans and mouse. Analysis of these genes and those from other species has reinforced our original proposal that these genes are part of a rapidly evolving gene family that share both structural and genomic similarities. Although it is now clear that some of the original ‘rules’ that we suggested as requirements for membership of this family do not hold true in all cases, it can been seen that, in general, all members of the family share many characteristics. Where divergence does occur, it is likely that this is the result of advantageous alterations to the proteins that will undoubtedly confer unique functions on them. It is likely that analysis of these divergent family members may aid in our understanding of the function of BPIF-containing proteins in general.

Proteins with a BPI/LBP/PLUNC-Like Domain: Revisiting the Old and Characterizing the New: A Biochemical Society Focused Meeting held at New Business School, University of Nottingham, U.K., 5–7 January 2011. Organized and Edited by Colin Bingle (Sheffield, U.K.) and Sven-Ulrik Gorr (University of Minnesota School of Dentistry, Minneapolis, MN, U.S.A.).

Abbreviations

     
  • BASE

    breast cancer and salivary gland expression

  •  
  • BPI

    bactericidal/permeability-increasing protein

  •  
  • BPIF

    BPI fold-containing

  •  
  • CETP

    cholesteryl ester-transfer protein

  •  
  • EST

    expressed sequence tag

  •  
  • LBP

    lipopolysaccharide-binding protein

  •  
  • PLTP

    phospholipid-transfer protein

  •  
  • PLUNC

    palate, lung and nasal epithelium clone

  •  
  • LPLUNC

    long PLUNC isoform

  •  
  • SPLUNC

    short PLUNC isoform

  •  
  • Vom

    vomeromodulin

Funding

This work is funded by the Wellcome Trust [grant number 107726], the British Lung Foundation and Sheffield Hospitals Charitable Trust.

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