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. Author manuscript; available in PMC: 2014 Dec 1.
Published in final edited form as: Gene. 2012 Oct 12;531(2):126–132. doi: 10.1016/j.gene.2012.09.111

Genetic complexity of the human surfactant-associated proteins SP-A1 and SP-A2

Patricia Silveyra 1, Joanna Floros 1,2
PMCID: PMC3570704  NIHMSID: NIHMS414897  PMID: 23069847

Abstract

Pulmonary surfactant protein A (SP-A) plays a key role in innate lung host defense, in surfactant-related functions, and in parturition. In the course of evolution, the genetic complexity of SP-A has increased, particularly in the regulatory regions (i.e. promoter, untranslated regions). Although most species have a single SP-A gene, two genes encode SP-A in humans and primates (SFTPA1and SFTPA2). This may account for the multiple functions attributed to human SP-A, as well as the regulatory complexity of its expression by a relatively diverse set of protein and non-protein cellular factors. The interplay between enhancer cis-acting DNA sequences and trans-acting proteins that recognize these DNA elements is essential for gene regulation, primarily at the transcription initiation level. Furthermore, regulation at the mRNA level is essential to ensure proper physiological levels of SP-A under different conditions. To date, numerous studies have shown significant complexity of the regulation of SP-A expression at different levels, including transcription, splicing, mRNA decay, and translation. A number of trans-acting factors have also been described to play a role in the control of SP-A expression. The aim of this report is to describe the genetic complexity of the SFTPA1 and SFTPA2 genes, as well as to review regulatory mechanisms that control SP-A expression in humans and other animal species.

Keywords: surfactant proteins, innate immunity, genetic variants, transcriptional regulation, evolution

1. Introduction

Pulmonary surfactant is a lipoprotein complex essential for life, as it prevents alveolar collapse at low lung volumes. In addition to maintaining alveolar integrity, surfactant plays an important role in lung host defense, and the control of inflammation, primarily by specific functions of the protein component (i.e. surfactant proteins). The genetic variability of surfactant proteins has been extensively studied, and variants have been associated with acute and chronic lung disease throughout life in a variety of population studies. The roles of the hydrophilic surfactant proteins (SP-A and SP-D) as innate immunity molecules and inflammatory mediators have also been extensively reported. We have recently reviewed the current knowledge of SP-A and SP-D genetic variations in pulmonary disease and pathogenesis (Silveyra and Floros, 2012b). In the present review, we focus on the human SP-A genes, their variants, and regulatory regions. We present an overview of the known regulatory mechanisms involved in SP-A gene expression at the genetic and epigenetic level, as well as the experimental models used to study this regulation. Moreover, we discuss evolutionary aspects of this regulation by comparing SP-A regulatory regions in humans and other animal species.

2. Pulmonary Surfactant

In the lung alveolar epithelium, type II pneumocytes synthesize and secrete a lipoprotein complex essential for life known as pulmonary surfactant. The main function of this complex is to reduce surface tension at the alveolar air-liquid interface, and consequently prevent alveolar collapse at low lung volumes. The composition of surfactant is approximately 90–92% lipids, and 8–10% proteins (Postle et al., 2001; Perez-Gil and Weaver, 2010). The lipid fraction is primarily phospholipids, the key components of the surface tension lowering function. The protein component participates in surfactant functions, as well as in the modulation of the innate immune response, and the regulation of inflammatory processes (Crouch, 1998; Crouch et al., 2000; Phelps, 2001). Surfactant homeostasis is not only critical for breathing (and thus survival) in the prematurely born infant, but also for maintaining lung health, and normal lung function throughout life, as quantitative and/or qualitative derangement in surfactant composition and/or function are associated with respiratory diseases (Floros and Phelps, 1997; Floros and Kala, 1998; Floros and Wang, 2001; Whitsett et al., 2010).

3. Surfactant proteins

Approximately 8–10% of pulmonary surfactant is composed by proteins. Surfactant-associated proteins are classified into two groups, based on their hydrophobicity properties. The hydrophobic, surfactant proteins B (SP-B), and C (SP-C) are primarily involved in the prevention of alveolar collapse. SP-B expression is altered in a number of acute and chronic lung diseases and absence of SP-B is not compatible with life, whereas SP-C stabilizes surfactant at low lung volumes, and plays a role in innate immunity (Wang et al., 1996; deMello and Lin, 2001; Glasser et al., 2001; Augusto et al., 2003; Nogee, 2004; Whitsett et al., 2010). The remaining two proteins, SP-A and SP-D, are hydrophilic proteins that belong to the C-type lectin family (collectins), and are primarily host defense proteins (Crouch and Wright, 2001; Hawgood and Poulain, 2001; Phelps, 2001; Floros et al., 2009). Members of the collectin family are characterized by an N-terminal collagen-like domain and a C-terminal carbohydrate recognition domain (CRD) that allows binding to various types of macromolecules, pathogens and allergens (Crouch and Wright, 2001). SP-A and SP-D are found in large oligomeric structures that have the ability to bind and opsonize several pathogens and allergens (Kishore et al., 2005; Kishore et al., 2006). SP-A and SP-D also play a role in surfactant homeostasis (Botas et al., 1998; Ikegami et al., 2000; LeVine et al., 2000; Hawgood and Poulain, 2001), and alterations of the levels of these two proteins have been reported in several pulmonary pathologies (Silveyra and Floros, 2012b).

3.1. Surfactant protein A

3.1.1. Function and structure

SP-A, the most abundant protein of surfactant, is involved in both host defense and surfactant-related functions. The role of SP-A in innate immunity is primarily mediated by its ability to bind several pathogens, enhance phagocytosis and chemotaxis of alveolar macrophages, induce proliferation of immune cells, and stimulate proinflammatory cytokine production, as well as to modulate the generation of reactive oxygen species (reviewed in (Phelps, 2001; Wright, 2005; Wang and Reid, 2007)). Furthermore, SP-A participates in several other functions that involve secretion of surfactant, signaling the initiation of parturition, as well as maintaining the structure of the extracellular form of surfactant tubular myelin (Williams et al., 1991; Condon et al., 2004; Wang et al., 2010; Snegovskikh et al., 2011; Yadav et al., 2011). Moreover, the expression and function of SP-A is not restricted to alveolar epithelial cells. Several studies have reported SP-A expression in the lower and higher airway epithelial cells, as well as extrapulmonary tissues including the lacrimal system (Bräuer et al., 2007), parotid glands (Bräuer et al., 2009), gingiva (Bräuer et al., 2012), epithelial cells of small and large intestine (Rubio et al., 1995) and Eustachian tube epithelium (Paananen et al., 2001), and vagina (MacNeill et al., 2004).

The structure of SP-A consists of four domains: an N-terminal, a collagen-like domain, a neck region, and a carbohydrate recognition domain (CRD) (Floros, 2001; Floros et al., 2009). The most conserved domains are the neck and N-terminal. The CRD consists of 115 amino acids, including four cysteine residues that are conserved across species. These form two pairs of disulfide bonds within the CRD that play a critical role in the stability of the SP-A structure (Floros et al., 2009). A cysteine residue located near the N-terminal (position 6 of the mature protein) has been proposed to participate in the supratrimeric assembly of SP-A (Sánchez-Barbero et al., 2007). Furthermore, the human and baboon SP-A1 gene contain an extra cysteine (Cys85) that may generate additional intermolecular disulfide bonds that affect SP-A1 structure, function, and biochemical properties (Wang et al., 2007a).

3.1.2. Human SFTPA1 and SFTPA2

The majority of species studied to date have a single SP-A gene. However, in primates and humans there are two functional genes (SFTPA1 and SFTPA2). In humans, the mature SP-A monomer is a 35kDa protein composed by 248 amino acids, and the two gene products (SP-A1 and SP-A2) differ in four amino acids at the coding region (Floros and Hoover, 1998; DiAngelo et al., 1999). The amino acid differences that distinguish between SP-A1 and SP-A2 genes and among their corresponding variants are located in the collagen-like domain. These are Met66, Asp 73, Ile81 and Cys85 for SP-A1, and Thr66, Asn73, Val81, and Arg85 for SP-A2. One of these has shown to affect its structure and function (Wang et al., 2007a). Furthermore, nucleotide differences that do or do not change the encoded amino acid among SFTPA1 or SFTPA2 variants are located within the sequence for the signal peptide, collagen-like region, and CRD (Floros, 2005; Floros et al., 2009). Several studies have identified functional differences between SP-A1 and SP-A2 in a variety of innate immunity and surfactant related functions including cytokine production (Wang et al., 2000; Wang et al., 2002; Huang et al., 2004), modulation of surfactant secretion (Wang et al., 2004), phagocytosis by alveolar macrophages (Mikerov et al., 2005; Mikerov et al., 2007; Mikerov et al., 2008a), as well as other structural characteristics (Garcia-Verdugo et al., 2002; Oberley and Snyder, 2003; Wang et al., 2007a; Wang et al., 2007b). However, no differences were found between SP-A1 and SP-A2 in the inhibition of hemagglutination activity of influenza A virus (Mikerov et al., 2008a). Moreover, functional divergence between SP-A1 and SP-A2 has been shown for an extracellular form of surfactant, the tubular myelin (Wang et al., 2010). Whether further functional divergence exists among SP-A1 and SP-A2 variants remains to be determined. Differences in structure, and posttranslational modifications have been also described between SP-A1 and SP-A2 (Floros et al., 2009), and some of the structural differences between SP-A1 and SP-A2 may be responsible for functional differences (Wang et al., 2007a). Moreover, differences in the expression of SFTPA1 and SFTPA2, as assessed by the protein ratio of SP-A1 to total SP-A, have been observed in human bronchoalveolar lavage samples as a function of age and lung health status (e.g. healthy vs. cystic fibrosis, culture positive vs. negative; asthmatic vs. control) (Tagaram et al., 2007; Wang et al., 2011).

SP-A is found in a variety of oligomeric structures. A 630 kDa hexameric bouquet-like structure contains a total of eighteen SP-A1 and SP-A2 monomers composed of six trimeric structural subunits of 105kDa. Both hetero-oligomers (i.e. consisting of both SP-A1, and SP-A2 monomers), and homo-oligomers (i.e. consisting of SP-A1 or SP-A2 monomers) are functional, and both gene products are required for tubular myelin formation (Voss et al., 1991; Wang et al., 2002; Mikerov et al., 2005; Mikerov et al., 2007; Wang et al., 2007a; Mikerov et al., 2008a; Wang et al., 2010). Differences between SP-A1 and SP-A2 in oligomerization properties, aggregation, structural stability, sugar-binding capacity, and ability to form phospholipids monolayers and tubular myelin have also been observed (Wang et al., 2000; Garcia-Verdugo et al., 2002; Wang et al., 2002; Oberley and Snyder, 2003; Wang et al., 2004; Mikerov et al., 2007; Sánchez-Barbero et al., 2007; Wang et al., 2007a; Wang et al., 2007b; Wang et al., 2010).

3.1.3. SP-A variants

More than 30 variants or intragenic haplotypes have been identified and characterized for SFTPA1 and SFTPA2 based on various combinations of single nucleotide polymorphisms (SNPs) within the coding region that may or may not change the encoded amino acids (Karinch and Floros, 1995a; DiAngelo et al., 1999; Floros, 2001; Floros and Wang, 2001; Floros et al., 2009). Among SFTPA1 variants, SNPs are found in the codons of amino acids 19, 50, 62, 133, and 219. Two of these polymorphisms are silent (amino acids 62 and 133), and the rest result in amino acid substitutions. Similarly, among SFTPA2 variants, SNPs are found in four codons: three silent (amino acids 9, 91, and 223) and one synonymous (amino acid 140) (DiAngelo et al., 1999). Of the variants identified, four SFTPA1 (6A, 6A2, 6A3, 6A4), and six SFTPA2 (1A, 1A0, 1A1, 1A2, 1A3, 1A5) variants have been observed in higher frequency in the general population (DiAngelo et al., 1999; Floros, 2001; Silveyra and Floros, 2012b). Furthermore, the frequency of these variants has been found to be variable in the population (DiAngelo et al., 1999; Floros, 2001; Liu et al., 2003).

3.1.4. SP-A receptors

As a critical component of innate immunity, SP-A helps combat infections caused by bacteria, viruses, fungi, and other pathogens by mechanisms that involve binding, aggregation, agglutination, inhibition of growth, and promotion of phagocytosis by activation of alveolar macrophages, an important cellular component of the lung first line of defense (McNeely and Coonrod, 1994; Schagat et al., 2001; Wu et al., 2003; Ding et al., 2004; Mikerov et al., 2007; Mikerov et al., 2008b; Mikerov et al., 2008c). SP-A can either bind pathogens to promote their destruction (opsonization), and/or activate immune cells by direct binding (Haagsman, 1998; LeVine and Whitsett, 2001; Wright, 2005), and/or stimulate the production of pro- and anti-inflammatory cytokines (Wang and Reid, 2007). Soluble receptors, as well as membrane receptors in the surface of alveolar macrophages have been shown to interact with SP-A. These include: CD35 (CR1), C1qR (CD93), CD14, CD91/calreticulin, SIRPα, SP-R210, gp-340, P63, TLR-2 and TLR-4, and others (reviewed in (Kishore et al., 2006; Bates, 2010; Silveyra and Floros, 2012b)). Some of these have been identified in the cell surface of alveolar type II pneumocytes, but not in alveolar macrophages, indicating that they may play a role in surfactant function, whereas others have been found to be ubiquitous.

3.2. Human SP-A Gene structure

The genomic locus of the human SP-A genes is located in the long arm of chromosome 10 (Bruns et al., 1987; Floros and Hoover, 1998), and it consists of the two functional genes, SFTPA1 and SFTPA2, and a pseudogene (SFTPA3P) (Hoover and Floros, 1998). The two functional genes are located in opposite transcriptional orientation in 10q22.3 (GenBank, NC_000010.10) as shown in Figure 1, and are in linkage disequilibrium (Liu et al., 2003). According to GenBank, the SFTPA1 genomic sequence (Gene ID: 653509) is located between positions 81370695-81375199 of the positive strand, whereas the SFTPA2 (Gene ID: 729238) is located in the complement strand, positions 81315608-81320163. The SFTPA3P pseudogene (Gene ID: 100288405) is also located in the complement strand, between positions 81353703-81355415. Similarly, the Ensembl database locates SFTPA1 between positions 81370701 81375202 of the positive strand (ENSG00000225827), and the pseudogene between positions 81355416-81355050 of the complement strand (ENSG00000225827). The SFTPA2 gene location is the same for both GenBank and Ensembl (ENSG00000185303). While both human SFTPA1 and SFTPA2 genes are approximately 4.5kb in length, and share approximately 94% nucleotide identity, their protein products share approximately 96% amino acid sequence identity. The structure of the two human genes is similar and consists of four coding exons (I–IV), and several untranslated exons (A, B, B′, C, C′,D, D′) (Karinch and Floros, 1995a) at 5′UTR as illustrated in Figure 1 (reviewed in (Floros, 2001; Floros and Wang, 2001; Floros, 2005; Floros et al., 2009)).

Figure 1.

Figure 1

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Upper panel Diagrammatic representation of the 10q22–23 locus of the human chromosome 10. The two human SP-A genes SFTPA1 and SFTPA2 are located in opposite transcriptional orientation, flanking a pseudogene (SFTPA3P). Lower panel. SP-A1 and SP-A2 common gene structure. Coding exons are indicated with numbers, and untranslated exons are indicated with letters. A* represents exons A, A′, and A″.

In general, the genetic complexity of SP-A has been shown to increase as one ascends the mammalian evolutionary ladder. Almost all mammalian species have been shown to contain a single SP-A gene, except from baboons and humans that have two. A gene duplication event occurred more than 26.5 million years ago (Gao et al., 1996), resulting in the generation of SFTPA1 and SFTPA2. Additional SP-A sequences have been reported in some species, e.g. three genes in opossum (SP-A1, SP-A2, and SP-A3) and two variants in chicken (SP-A and an SP-A-like gene) (Gao et al., 1996; Hogenkamp et al., 2006; Hughes, 2007). All SP-A genes of various species are about 5 Kb in length, with the basic exon-intron structure being conserved in all species studied so far. The sequence similarity among species is higher in exons than in introns. The human and baboon genes contain additional exons/introns at the 5′ untranslated region that are alternatively spliced generating mRNA splice variants (Karinch and Floros, 1995a; Gao et al., 1996). Furthermore, in humans, splice and sequence differences between the functional genes and/or their variants give rise to a number of variable transcripts, that have been shown to differentially affect SP-A gene expression (Karinch and Floros, 1995a; Karinch et al., 1998; Wang et al., 2003; Wang et al., 2005; Wang et al., 2009; Silveyra et al., 2010; Silveyra et al., 2011). This increase in genetic complexity, in particular at the regulatory regions, may reflect the functional importance of SP-A during evolution, and the necessity to control protein levels in specific physiological or pathological situations (Mendelson et al., 1998; Floros, 2005; Tagaram et al., 2007; Bruce et al., 2009; Vlachaki et al., 2010; Wang et al., 2011; Silveyra and Floros, 2012b).

3.3. SP-A promoter and control of transcription

To date, several regulatory regions have been identified within the 5′ flanking regions and the human SFTPA1 and SFTPA2 gene sequences and in particular in the first 300 base pairs of the proximal promoter, immediately upstream of the transcription start site. These include, cAMP response elements (CRE), and GT-boxes important for high basal and c-AMP mediated regulation of SFTPA2 in alveolar epithelial cells (Young and Mendelson, 1996; Young and Mendelson, 1997), as well as TTF-1 binding elements (TBE), important for hormonal regulation of lung development, as well as temporal and spatial control of SP-A gene expression (Yi et al., 2002; Liu et al., 2008). Some of these elements are conserved among species. A number of lung cell-specific transcription factors and small molecules have been shown to interact with regulatory sequences present at the human SP-A promoters, in particular during the late stages of development, where the expression of SP-A transitions from practically silent to dramatically upregulated (Gao et al., 2003; Alcorn et al., 2004; Liu et al., 2008). Some of the molecules shown to affect SP-A expression include: estrogen receptor alpha (Liu et al., 2006), dexamethasone (Hoover et al., 1999; Alcorn et al., 2004; Islam and Mendelson, 2008), NfkB (Islam and Mendelson, 2002; Wu et al., 2011), C/EBP (Rosenberg et al., 2002), phorbol esters (Hoover et al., 2000), c-AMP (Young and Mendelson, 1996; Young and Mendelson, 1997; Liu et al., 2009), TTF-1 (Li et al., 1998; Alcorn et al., 2004; Liu et al., 2008), and others (Karinch et al., 1998; Kumar and Snyder, 998). Several of these factors, in addition to modulating the expression of SP-A in humans, affect the expression of rabbit, murine, and baboon SP-A genes (Lacaze-Masmonteil et al., 1992; Alcorn et al., 1993; Kouretas et al., 1993; Bruno et al., 1995; Rosenberg et al., 1999; Bruno et al., 2000; Rosenberg et al., 2002).

In addition to these sequence-specific interacting factors at the SP-A promoters, another level of complexity appears to play a role in the control of expression of human SP-A genes, known as epigenetic modifications. Epigenetic modifications have been shown to affect the expression of SP-A1 and SP-A2 mRNAs in a variety of physiological and pathological conditions. Recent unpublished data from our group identified significant differences in the methylation status of specific cytosines of the SFTPA2 5′ flanking region between normal and adenocarcinoma human paired samples. The hypermethylated DNA in cancerous tissue correlated with decreased SP-A2 mRNA expression. These findings may hold promise for future use of SP-A2 as a biomarker for the diagnosis and/or therapies of lung cancer. Moreover, previous studies from our group reported associations of altered methylation patterns of CpG sites in the human SFTPA1 promoter with lung cancer (Lin et al., 2007). Furthermore, histone acetylation and methylation at regulatory regions of the SP-A gene promoters have been shown to affect SP-A expression in lung cells during development and during hypoxia (Islam and Mendelson, 2006; Lin et al., 2007; Islam and Mendelson, 2008; Vaid and Floros, 2009; Benlhabib and Mendelson, 2011; Silveyra and Floros, 2012a).

In summary, gene regulation is a dynamic and complex process. The promoter region of SP-A may serve as an enhanceosome (Benlhabib and Mendelson, 2011) through which SP-A expression is modulated via the interaction of several transcription factors with specific sequence elements, some of which are subject to epigenetic regulation, and may be modulated by pathological conditions (e.g. cancer) and environmental insults (Silveyra and Floros, 2012a). Given the functional differences between SFTPA1 and SFTPA2 and their genetic complexity, differences in the regulation of expression among SP-A variants may underlie individual susceptibility in response to different insults such as oxidative stress and disease (Mendelson et al., 1998; Tagaram et al., 2007; Floros et al., 2009; Wang et al., 2011).

3.4. SP-A mRNA

Transcription of SFTPA1 and SFTPA2 genes results in a variety of mRNA transcripts. The source of this variability involves differences in the transcription start sites, as well as alternative splicing of exons (A, B, B′, C, C′, D, D′) at the 5′UTR. The formation of SP-A 5′UTR splice variants is not a random process as there are major, minor, and rare splice-variants for SP-A1 and SP-A2 transcripts (Karinch and Floros, 1995a). Furthermore, alternative transcription start sites have been observed for SFTPA1 and SFTPA2 by primer extension in two independent studies (McCormick et al., 1994; Karinch and Floros, 1995a); these identified three transcription start sites for SFTPA1, and one common transcript start site for SFTPA2, although some minor discrepancies observed between the two studies were attributed to variations in the individual lung RNA samples, and/or the limited number of clones tested (Floros et al., 2009).

The predominant human SP-A transcript splice variants and the frequencies with which they are found differ between the two genes. Similarly, frequency differences may also exist among individuals for these mRNAs (Karinch and Floros, 1995b). For SP-A1, the major splice variant is AD′ (81%), followed by ACD′ and AB′D′ (7%), and for SP-A2 the major variants are ABD′ (49%), and ABD (44%) (Karinch and Floros, 1995a). Although more than 30 variants for human SFTPA1and SFTPA2 have been identified in the population, the sequence for those found in the population with higher frequencies is available in GenBank under the accession numbers indicated in Table 1.

Table 1.

Gen Bank sequences for SP-A1 and SP-A2 mRNA transcripts

Gene Variant id 5′UTR splice Coding 3′JTR sequence GenBank id
SP-A1 AD′6A AD′ 6A 6A HQ021433
SP-A1 AD′6A2 AD′ 6A2 6A2 HQ021434
SP-A1 AD′6A3 AD′ 6A3 6A3 HQ021435
SP-A1 AD′6A4 AD′ 6A4 6A4 HQ021436
SP-A1 AB′D′6A AB′D′ 6A 6A JX502764
SP-A1 AB′D′6A2 AB′D′ 6A2 6A2 HQ021437
SP-A1 AB′D′6A3 AB′D′ 6A3 6A3 HQ021435
SP-A1 AB′D′6A4 AB′D′ 6A4 6A4 HQ021439
SP-A1 ACD′6A ACD′ 6A 6A JX502765
SP-A1 ACD′6A2 ACD′ 6A2 6A2 HQ021440
SP-A1 ACD′6A3 ACD′ 6A3 6A3 HQ021441
SP-A1 ACD′6A4 ACD′ 6A4 6A4 HQ021442
SP-A2 ABD1A ABD 1A 1A HQ021432
SP-A2 ABD1A0 ABD 1A0 1A0 HQ021421
SP-A2 ABD1A1 ABD 1A1 1A1 HQ021422
SP-A2 ABD1A2 ABD 1A2 1A2 HQ021423
SP-A2 ABD1A3 ABD 1A3 1A3 HQ021424
SP-A2 ABD1A5 ABD 1A5 1A5 HQ021425
SP-A2 ABD′1A ABD′ 1A 1A HQ021426
SP-A2 ABD′1A0 ABD′ 1A0 1A0 HQ021427
SP-A2 ABD′1A1 ABD′ 1A1 1A1 HQ021423
SP-A2 ABD′1A2 ABD′ 1A2 1A2 HQ021429
SP-A2 ABD′1A3 ABD′ 1A3 1A3 HQ021430
SP-A2 ABD′1A5 ABD′ 1A5 1A5 HQ021431
SP-A1 variant 1 AB′D′ 6A3 6A3 NM_005411.4
SP-A1 variant 2 ACD′ 6A3 6A3 NM_001093770.2
SP-A1 variant 3 ABD′ 6A3 6A3 NM_001164644.1
SP-A1 variant 4 AD′ 6A3 6A3 NM_001164647.1
SP-A1 variant 5 ACD′ 6A3 (truncated)a 6A3 NM_001164645.1
SP-A1 variant 6 AB′D′ 6A3 (truncated)a 6A3 NM_001164646.1
SP-A2 ABD′ 1A2 1A0 NM_001098668.2
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a

missing a fragment of 147 nt (positions 238–435 mRNA; positions 84–231 from start codon)

SP-A1 and SP-A2 mRNA variants also show sequence differences at the 3′UTRs (Krizkova et al., 1994; Hoover and Floros, 1999; Wang et al., 2003; Silveyra et al., 2010). These, together with several other mRNA elements, such as the 5′CAP, poly(A) tail, secondary structures, and other structural elements, may modulate mRNA translation and thus differentially control SP-A1 and SP-A2 protein levels.

3.4.1 Role of SP-A UTRs in the control of transcription and translation

Several studies from our laboratory have reported that SP-A 5′UTR splice variants affect gene expression with variable efficiency, and that variants containing the untranslated exon B have an increased rate of transcription, and translation in both in vitro (Silveyra et al., 2010) and in lung epithelial cell culture systems (Wang et al., 2005; Silveyra et al., 2010). When compared to SP-A1 5′UTR variants ACD′, AD′, and AB′D′, the SP-A2 5′UTR variant ABD displayed a significantly lower rate of mRNA decay and a higher translation efficiency (Wang et al., 2005), and postulated that alternative splicing at the SP-A 5′UTR is a major regulatory mechanism for differential SP-A1 and/or SP-A2 variant expression (Wang et al., 2005; Silveyra et al., 2010; Silveyra et al., 2011). In support of this, our preliminary studies in which lung cells were exposed to diesel exhaust particulate matter showed that the splicing pattern of SP-A 5′UTRs is altered (our unpublished data), and the efficiency of SP-A translation is increased in variants containing exon B (Wang et al., 2009). Furthermore, the SP-A2 5′UTRs that contain exon B (absent in SP-A1 variants) has been shown, via a specific double-loop secondary structure, to form an internal ribosomal entry site (IRES) and mediate cap-independent translation, a process that often occurs under cellular stress, hypoxia, and other insults (Wang et al., 2009; Komar and Hatzoglou, 2011). Moreover, infectious agents such as respiratory syncytial virus also affect the rate of SP-A translation in pulmonary epithelial cells, although the mechanisms involved in this inhibition are still unknown (Bruce et al., 2009).

A recent study identified potential regulatory sequence elements in the 30 nucleotide SP-A2 untranslated exon B that may modulate gene expression via interaction with trans-acting factors. The exon B DNA sequence was shown to exhibit characteristics of a transcriptional enhancer (Silveyra et al., 2011), and at the mRNA level, exon B enhanced translation when placed in its natural sequence environment (ABD) or within orthologous sequences (Silveyra et al., 2011). The enhancing effect of exon B in SP-A expression may involve sequence motifs and/or secondary structures that potentially interact with specific RNA-binding factors and modulate a variety of post-transcriptional events. Preliminary results from our group using RNA gel shift assays identified several exon B binding proteins, and mapped specific nucleotides of exon B important for these interactions (our unpublished data). Moreover, the splice variant ACD′, found in SP-A1 transcripts (but not in SP-A2 transcripts), contains two upstream AUG codons (uAUGs) at its unique untranslated exon C that may affect translational efficiency. One of these is in frame with the main start codon (Karinch and Floros, 1995b), and shown to be functional (our preliminary data).

Variant-specific sequences located in the SP-A 3′UTRs may differentially regulate SP-A expression at the translational level. A pyrimidine-rich domain of 37 nucleotides is present in the 3′UTRs of SP-A1 and SP-A2 variants, but 7 of the nucleotides in this region differ between SP-A1 and SP-A2. Moreover, the 3′-UTRs of SP-A2 variants contain two AU-rich elements (AUUUA), whereas the SP-A1 sequences contain only one. The published literature indicates that these elements may influence mRNA stability, as well as the regulation of SP-A1 and SP-A2 variants by glucocorticoids (Hoover and Floros, 1999; Wang et al., 2003). An 11-nt element, present in the 3′UTR of all the SP-A2 variants and the SP-A1 variant 6A2 has been shown to negatively affect translation efficiency, by mechanisms that involve formation of secondary structures with different stabilities, as well as differences in predicted miRNA binding to this element (Silveyra et al., 2010). Interestingly, the effects observed were more evident when the transcripts were polyadenylated, indicating a potential role of the poly(A) tail in the control of translation of SP-A transcripts (Silveyra et al., 2010; Silveyra et al., 2011). Given the nucleotide differences among SP-A variants at the 3′UTR, it is highly likely that differences observed in their translational regulation are mediated by miRNAs. Our preliminary studies in cell lines expressing different SP-A variants indicate a role of specific miRNAs in the control of SP-A translation (our unpublished data).

In summary, the UTRs of SP-A mRNA transcripts are implicated in the control of SP-A expression. Several gene-specific, and variant-specific elements have been described and studied in these regions. It is possible that the ability of the variants to bind regulatory factors at the UTRs differs, and that this may account for differential protein expression of SP-A1 and SP-A2 variants under normal or compromised conditions (i.e. lung disease).

4. Genetic associations of SP-A variants with lung disease

The influence of genetic variants in acute and chronic lung disease susceptibility have been studied within different biological contexts, and correlated with environmental factors, and/or other conditions including prematurity, concurrent diseases, or need for mechanical ventilation (Leikauf et al., 2002; Hallman and Haataja, 2003; Villar et al., 2003; Christie, 2004; Grigoryev et al., 2004; Kishore et al., 2005; Nonas et al., 2005; Meyer and Garcia, 2007; Lam and dos Santos, 2008; Reddy and Kleeberger, 2009; Meyers, 2010). Numerous associations between SFTPA1 and SFTPA2 genetic variants and acute and chronic lung disease have been identified in several populations and study groups including neonates, children, and adults. For example, the SFTPA2 1A3 variant has been associated with susceptibility to tuberculosis in both Mexican and Ethiopians (Floros et al., 2000; Malik et al., 2006). In neonates, a specific SFTPA1/SFTPA2 haplotype, 6A2/1A0, has been shown to associate with risk for respiratory distress syndrome (RDS) in Finnish and non-Finnish Caucasians (Rämet et al., 2000; Floros et al., 2001). RDS is a condition where insufficient amounts of surfactant proteins have been observed, as well as absence of the extracellular form of surfactant, the tubular myelin, where SP-A is an essential component. The SFTPA1 and SFTPA2 genetic associations have been reviewed elsewhere (Floros and Kala, 1998; Floros and Pavlovic, 2003; Floros and Thomas, 2009; Silveyra and Floros, 2012b).

5. Concluding remarks

Human SP-A expression is controlled developmentally and by tissue specificity at both transcriptional and translational levels. The SP-A 5′ and 3′UTRs contain several regulatory elements implicated in the differential regulation of SP-A1 and SP-A2 expression, as well as their genetic variants. Study of mechanisms involved in these processes may help explain the altered ratio of SP-A1 and SP-A2 protein content in certain lung diseases or conditions (Tagaram et al., 2007; Wang et al., 2011), as well as gain insight into the basis of individual disease susceptibility.

Although there is considerable evidence that the human SP-A complexity is associated with various pulmonary diseases, and that differences in the function and structure of SP-A1 and SP-A2 exist as well as differences in the relative levels of SP-A1 to total SP-A in several diseases, the underlying mechanisms are not yet entirely known. We postulate that given the functional differences between SP-A1 and SP-A2, the overall SP-A functional activity in the lung depends on the relative levels of SP-A1 and SP-A2 rather than the total SP-A content (i.e., without regard to the SP-A1 and SP-A2 proportions), and that an altered regulation of SP-A1 or SP-A2 gene expression could result in an unfavorable SP-A1 to SP-A2 ratio for normal host defense.

Highlights.

  • Structure of surfactant protein A genes

  • Characteristics of SP-A1 and SP-A2 variants

  • Regulatory aspects of SP-A1 and SP-A2 expression

Acknowledgments

This work was supported by NIH-HL34788.

Footnotes

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