Characterization of a human surfactant protein A1 (SP-A1) gene-specific antibody; SP-A1 content variation among individuals of varying age and pulmonary health

Tagaram, Hephzibah Rani S.; Wang, Guirong; Umstead, Todd M.; Mikerov, Anatoly N.; Thomas, Neal J.; Graff, Gavin R.; Hess, Joseph; Thomassen, Mary Jane; Kavuru, Mani S.; Phelps, David S.; Floros, Joanna

doi:10.1152/ajplung.00249.2006

Cited by 60 publications

(88 citation statements)

References 81 publications

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“…Variations of the ratio of SP-A1 to total SP-A protein content have been observed as a function of pulmonary disease and aging (44). Therefore, the relative levels of SP-A1 and SP-A2 proteins could relate to disease susceptibility and/or function as predictors of disease specificity.…”

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confidence: 99%

“…On the basis of these findings, it was hypothesized that eB is a cis-acting regulatory element for both transcription and translation (42,48,50). Moreover, eB confers SP-A 5=UTR variants with differential capabilities of modulating expression, and this in turn may contribute to differences observed in SP-A levels among individuals under several conditions (e.g., lung disease, aging) (44,53). However, the location and properties of cisacting elements in the UTRs of SP-A1 and SP-A2 transcripts have not been described.…”

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confidence: 99%

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The untranslated exon B of human surfactant protein A2 mRNAs is an enhancer for transcription and translation

Silveyra

Raval²,

Simmons³

et al. 2011

American Journal of Physiology-Lung Cellular and Molecular Phys

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) and SFTPA2 (SP-A2), encode surfactant protein A, a molecule of innate immunity and surfactant-related functions. Several genetic variants have been identified for both genes. These include nucleotide (nt) polymorphisms, as well as alternative splicing patterns at the 5= untranslated region (5=UTR). Exon B (eB) is included in the 5=UTR of most SP-A2, but not SP-A1 splice variants. We investigated the role of eB in the regulation of gene expression and translation efficiency. A luciferase (Luc) reporter gene was cloned downstream of the entire (AeBD) or eB deletion mutants (del_mut) of the SP-A2 5=UTR, or heterologous 5=UTRs containing the eB sequence, or a random sequence of equal length. The del_mut constructs consisted in consecutive deletions of five nucleotides (n ϭ 8) within eB and the exon-exon junctions in the AeBD 5=UTR. Luc activities and mRNA levels were compared after transfection of NCI-H441 cells. We found that 1) eB increased Luc mRNA levels when placed upstream of heterologous 5=UTR sequences or the promoter region, regardless of its position and orientation; 2) translation efficiency of in vitrogenerated mRNAs containing eB was higher than that of mRNAs without eB; and 3) the integrity of eB sequence is crucial for transcription and translation of the reporter gene. Thus eB 1) is a transcription enhancer, because it increases mRNA content regardless of position and orientation, 2) enhances translation when placed in either orientation within its natural 5=UTR sequence and in heterologous 5=UTRs, and 3) contains potential regulatory elements for both transcription and translation. We conclude that eB sequence and length are determinants of transcription and translation efficiency. surfactant protein A; untranslated regions; secondary structure stability; trans-binding sites; cell-specific regulatory factors SURFACTANT PROTEIN A (SP-A) plays an important role in host defense and surfactant-related functions. Two functional genes, SFTPA1 (SP-A1), and SFTPA2 (SP-A2), encode this protein in humans. More than 30 gene variants have been described and characterized on the basis of nucleotide polymorphisms and amino acid sequences at the coding region (7,8,10). At the mRNA level, variants show differences at the untranslated regions (UTRs), resulting in sequence variants at the 3=UTR (16,23,42,49) and splice variants at the 5=UTR (20). The genomic sequence of SP-A 5=UTR contains several exons (A, B, B=, C, C=, D, D=) (Fig. 1A) that are alternatively spliced to give rise to specific SP-A1 and SP-A2 patterns. The formation of SP-A 5=UTR splice variants is not a random process since there are major, minor, and rare splice variants for SP-A1 and SP-A2 transcripts (20). A study of SP-A transcripts found that the predominant splice variant configurations and their frequencies differ between the two genes, and these likely also differ among individuals (21). The major splice variant for SP-A1 is AD= (81%), followed by ACD= and AB=D= (7%). The major SP-A2 variants are ABD= (49%), and ABD (44%) (20).In humans, se...

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confidence: 99%

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confidence: 99%

The untranslated exon B of human surfactant protein A2 mRNAs is an enhancer for transcription and translation

Silveyra

Raval²,

Simmons³

et al. 2011

American Journal of Physiology-Lung Cellular and Molecular Phys

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“…However, no differences have been observed between SP-A1 and SP-A2 in their ability to inhibit hemagglutination activity of influenza A virus (40). Quantitative differences include differences between the SP-A1 and SP-A2 genes and/or variants in basal mRNA levels and in response to dexamethasone (22,33,48,58) and in protein levels in bronchoalveolar lavage (BAL) fluids from different individuals (52). Mechanisms involving NF-B activation (21,28) and mRNA stability and translational control (57) may contribute to quantitative differences.…”

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confidence: 99%

“…It is possible that the increased sensitivity to infection that some individuals exhibit after ozone exposure, as assessed by higher risk of common infections in areas with highly polluted air (42), may reflect the involvement of intrinsic or genetic factors and/or the functional impairment of oxidant-exposed SP-A. Different individuals may possess different combinations of SP-A variants and proportions of SP-A1 and SP-A2 content (52). Because different SP-A variants have been identified with qualitative and quantitative differences, and functional differences between SP-A1 and SP-A2 have been observed (15,20,39,43,56,60,61), we speculate that the ratio of SP-A1 to SP-A2 is a better indicator of the overall ability of hSP-A to enhance phagocytosis in the lung than the total SP-A content.…”

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Impact of ozone exposure on the phagocytic activity of human surfactant protein A (SP-A) and SP-A variants

Mikerov¹,

Umstead²,

Gan³

et al. 2008

American Journal of Physiology-Lung Cellular and Molecular Phys

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Surfactant protein A (SP-A) enhances phagocytosis of Pseudomonas aeruginosa. SP-A1 and SP-A2 encode human (h) SP-A; SP-A2 products enhance phagocytosis more than SP-A1. Oxidation can affect SP-A function. We hypothesized that in vivo and in vitro ozone-induced oxidation of SP-A (as assessed by its carbonylation level) negatively affects its function in phagocytosis (as assessed by bacteria cell association). To test this, we used P. aeruginosa, rat alveolar macrophages (AMs), hSP-As with varying levels of in vivo (natural) oxidation, and ozone-exposed SP-A2 (1A, 1A 0 ) and SP-A1 (6A 2 , 6A 4 ) variants. SP-A oxidation levels (carbonylation) were measured; AMs were incubated with bacteria in the presence of SP-A, and the phagocytic index was calculated. We found: 1) the phagocytic activity of hSP-A is reduced with increasing levels of in vivo SP-A carbonylation; 2) in vitro ozone exposure of hSP-A decreases its function in a dose-dependent manner as well as its ability to enhance phagocytosis of either gram-negative or gram-positive bacteria; 3) the activity of both SP-A1 and SP-A2 decreases in response to in vitro ozone exposure of proteins with SP-A2 being affected more than SP-A1. We conclude that both in vivo and in vitro oxidative modifications of SP-A by carbonylation reduce its ability to enhance phagocytosis of bacteria and that the activity of SP-A2 is affected more by in vitro ozone-induced oxidation. We speculate that functional differences between SP-A1 and SP-A2 exist in vivo and that the redox status of the lung microenvironment differentially affects function of SP-A1 and SP-

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“…For instance, SP-A2 showed higher biological activity in inducing TNF␣ production by THP-1 cells (92,93), stimulating Pseudomonas aeruginosa association with rat alveolar macrophages (58,59) and inhibiting phosphatidylcholine secretion from alveolar type II cells (87). The relative amounts of SP-A1 and SP-A2 vary and seem to be affected by age and health status (81,94). The functional difference between the two products can probably be explained by structural and oligomerization differences (71,87,91).…”

Section: Discussionmentioning

confidence: 99%

Regulation of translation by upstream translation initiation codons of surfactant protein A1 splice variants

Tsotakos

Silveyra

Lin

et al. 2015

American Journal of Physiology-Lung Cellular and Molecular Phys

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Surfactant protein A (SP-A), a molecule with roles in lung innate immunity and surfactant-related functions, is encoded by two genes in humans: SFTPA1 (SP-A1) and SFTPA2 (SP-A2). The mRNAs from these genes differ in their 5′-untranslated regions (5′-UTR) due to differential splicing. The 5′-UTR variant ACD′ is exclusively found in transcripts of SP-A1, but not in those of SP-A2. Its unique exon C contains two upstream AUG codons (uAUGs) that may affect SP-A1 translation efficiency. The first uAUG (u1) is in frame with the primary start codon (p), but the second one (u2) is not. The purpose of this study was to assess the impact of uAUGs on SP-A1 expression. We employed RT-qPCR to determine the presence of exon C-containing SP-A1 transcripts in human RNA samples. We also used in vitro techniques including mutagenesis, reporter assays, and toeprinting analysis, as well as in silico analyses to determine the role of uAUGs. Exon C-containing mRNA is present in most human lung tissue samples and its expression can, under certain conditions, be regulated by factors such as dexamethasone or endotoxin. Mutating uAUGs resulted in increased luciferase activity. The mature protein size was not affected by the uAUGs, as shown by a combination of toeprint and in silico analysis for Kozak sequence, secondary structure, and signal peptide and in vitro translation in the presence of microsomes. In conclusion, alternative splicing may introduce uAUGs in SP-A1 transcripts, which in turn negatively affect SP-A1 translation, possibly affecting SP-A1/SP-A2 ratio, with potential for clinical implication.

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Characterization of a human surfactant protein A1 (SP-A1) gene-specific antibody; SP-A1 content variation among individuals of varying age and pulmonary health

Cited by 60 publications

References 81 publications

The untranslated exon B of human surfactant protein A2 mRNAs is an enhancer for transcription and translation

The untranslated exon B of human surfactant protein A2 mRNAs is an enhancer for transcription and translation

Impact of ozone exposure on the phagocytic activity of human surfactant protein A (SP-A) and SP-A variants

Regulation of translation by upstream translation initiation codons of surfactant protein A1 splice variants

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