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Allele-specific N-glycosylation delays human surfactant protein B secretion in
vitro and associates with decreased protein levels in vivo
ONLINE DATA SUPPLEMENT
Online Supplement: Supplemental Materials and Methods
Sample collection and study population
Sample pairs of amniotic fluid and umbilical cord blood (n = 273) were collected during 2000–2009
from elective Cesarean section deliveries from singleton pregnancies at the Oulu University
Hospital in Finland. This initial study population included fetuses of the Finnish parental origin
from pregnancies with gestation ranging from 30 to 40 completed weeks. The majority of the
samples were obtained from pregnancies resulting in deliveries at term (GA ≥ 37 weeks).
Information of the total volume of the amniotic fluid was not available. To obtain a relatively
homogenous study population, the preterm (GA < 37 weeks, n = 22) births were not included in the
genetic analyses to explore the correlation between relative SP-B levels and the SFTPB
polymorphisms, but were included to support the validity of the assay. The actual study population
for the genetic analysis included only sample pairs from term pregnancies (n = 251). In the
multivariate linear regression analyses, respiratory distress was defined as the diagnosis of RDS,
transient tachypnea of the newborn (TTN), or spontaneous pneumothorax. The diagnosis of RDS
was based on typical symptoms of respiratory distress shortly after birth or a requirement for
surfactant therapy, and on characteristic chest X-ray findings. TTN was diagnosed as the presence
of an acute respiratory distress in a term or near term infant, with radiographic findings consistent
with retained fetal lung fluid. The duration of TTN was between 24 and 48 hours and the
requirement of supplemental oxygen was less than 24 hours. The diagnosis of pneumothorax was
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determined by chest X-ray of infant with respiratory distress shortly after birth. Tension
pneumothorax was treated with a chest tube. Adult human lung tissue was obtained from patients
undergoing lung surgery. Informed consent was obtained from all of the subjects. The study was
approved by the Ethics Committee of the Oulu University Hospital.
Amniotic fluid sample preparation, western analysis and validity of the assay
Amniotic fluid specimens were frozen to −70°C shortly after collection and stored at this
temperature until analyzed. For the analyses, samples were thawed and cell debris was removed by
centrifugation at 100 x g. Proteins were separated under mildly reducing conditions (i.e. samples
were neither treated with β-mercaptoethanol nor heated, and the only reducing element in the
experiment was SDS), under which SP-B still remains as a dimer, in aliquots of 5 μl and 10 μl of
each amniotic fluid sample using 12% Bis-Tris polyacrylamide gels (Invitrogen, Carlsbad, CA) in
1× MES running buffer (Invitrogen). Similar aliquots of the same pooled amniotic fluid sample
known to contain high levels of SP-B were used as a standardized reference in each gel. Proteins
resolved by SDS-PAGE were transferred to PROTRAN nitrocellulose membranes (Whatman
GmbH, Dassel, Germany), which were probed using polyclonal rabbit anti-sheep SP-B (AB3780,
Chemicon, Temecula, CA) antibody diluted 1:10,000, followed by IRDye800-conjugated donkey
anti-rabbit IgG (611-732-127, Rockland Immunochemicals Inc., Gilbertsville, PA) diluted 1:3,000.
Direct fluorescence detection and quantitation of mature SP-B was performed using Odyssey
Infrared Imaging System 2.1 software (LI-COR Biosciences, Lincoln, NE). Densitometry was
performed using Quantity One software (Bio-Rad Laboratories Inc., Hercules, CA). Amniotic fluid
samples that were heavily contaminated with blood were excluded from the study (n = 10). Mature
dimeric SP-B was detected in every sample.
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The relative SP-B level for each sample was calculated by dividing the mean sample intensity (two
replicates) with the mean intensity of the standardized reference sample (two replicates) that was
used in the same experiment (western blot gel). The reference sample was given a value of 100 and
the other samples were given a value of their ratio to the reference sample multiplied by 100. For
example, a value of 200 in a study sample would thus mean a relative protein concentration of twice
as much as that in the reference sample.
Since the results of SP-B quantitation are critically dependent upon the assay used to measure the
protein levels, the validity of quantitation was tested by densitometric analysis of infra-red dyebased western immunoblot assay (more detailed information about the initial assay see
Supplemental Reference 1). The western quantitation assay was explored for reproducibility,
linearity, level of detection and sensitivity. Different volumes of the reference sample was used in
serial dilutions of 1:1, 1:2, 1:4, 1:8, and 1:16, representing sample volumes of 1.25; 2.5; 5, 10 and
20 µl, respectively. Our tests showed that the assay was repeatedly linear in the range of the sample
volumes used in the study. Although the method is not sensitive enough for accurate quantitation, it
can be concluded that its reproducibly yielded a constant response and a semi-quantitative estimate
of the relative protein levels.
To provide further support for the feasibility of the quantitation method, known clinical factors
(prematurity, respiratory distress) that might affect SP-B levels were studied. This was done using a
small set of samples from preterm births (n = 22), which were available beyond the actual study
population of the genetic analyses. This resulted in total of 273 sample pairs available from term
and preterm births. Relative SP-B protein concentrations in amniotic fluid specimens from fetuses
that developed RDS shortly after birth (n = 8) differed significantly from healthy controls (p <
0.001): the mean and median of SP-B protein levels were 6.6- and 7.9-fold higher, respectively, in
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healthy controls compared to the RDS group (Supplemental Figure S1). This result is consistent
with developmental SP-B deficiency, as expected based on preterm gestational age. SP-B protein
levels differed significantly between the TTN (n = 24) and healthy control groups (p < 0.001) with
mean/median SP-B levels 37.5/25.0 (SE 7.7) and 63.1/55.5 (SE 2.6), respectively. Controls tended
to have higher SP-B levels than infants with respiratory distress due to spontaneous pneumothorax,
but the number of affected infants (n = 3) was too low for adequate statistical power. All of the
available samples (n = 273) were included in these analyses described above. The findings of the
low relative SP-B concentrations in the samples associated with poor pulmonary outcomes (RDS
and TTN) supports the validity of the assay for relative quantitation.
The advantage of the western method over ELISA is that it allowed quantitating the correct mature
dimerized SP-B independently of the partially processed (25 kDa) or unprocessed (42 kDa) proSPB forms, which were also detected by the SP-B antibody. Western blot based quantitation was
therefore justified as the method of choice, and also the validation experiments showed the relative
protein concentration estimates to be precise enough and appropriate for the present study.
DNA sample preparation and genotyping of SFTPB gene polymorphisms
Genomic DNA was extracted from freshly frozen EDTA-anticoagulated umbilical cord blood
specimens with the Puregene DNA isolation kit (Qiagen, Hilden, Germany) and from lung tissue
samples using the Wizard Genomic DNA Purification kit (Promega, Madison, WI). Genotyping of
SFTPB Ile131Thr (rs1130866) polymorphism and Δi4 was performed as described previously
(Supplemental Reference 2). For the Δi4 polymorphism, the sizes of the resulting PCR fragments
were 240–660 bp, and the fragments smaller or larger than 513 bp (invariant or wild-type allele)
were defined as deletion or insertion variants, respectively.
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Genotyping of SFTPB polymorphisms C/A(−18) (rs2077079) and A/G(−384) (rs3024791) was
based on PCR and RFLP analysis using BseLI (Fermentas GmbH, Germany) for rs2077079 and
SmlI (New England Biolabs, Ipswich, MA) for rs3024791. For C/A(-18) primers 5′TAAGGCAGGCAGGAGTGTTC-3′
(forward)
and
5′-CACTGCAGCAGGTGTGACTC-3′
(reverse), followed by a nested amplification with primers 5′-AAGGCAGGCAGGAGTGTTCC-3′
(forward) and 5′-ACTGCAGCAGGTGTGACTCA-3′ (reverse), and for A/G(-384) mismatch
primers
forward
5′-CCAGGCAGGAAGCTATCAAG-3′
and
reverse
5′-
TCAAGCCTCCATACCTGGTC-3′ were used.
Reverse transcriptase PCR, construction of plasmids, and generation of stably transfected
proSP-B–expressing CHO cells
The RNA used in RT-PCR was extracted from human lung tissue heterozygous for SFTPB
Ile131Thr using TRI reagent (Sigma-Aldrich, St. Louis, MO). The Ile131Thr genotype was
determined from the corresponding lung DNA. The extracted RNA was further purified with the
RNeasy Micro kit (Qiagen) according to the manufacturer’s protocol. Three micrograms of purified
RNA was transcribed to cDNA using Moloney murine leukemia virus (M-MuLV) reverse
transcriptase (Promega). RT-PCR was performed using primers specific for the full-length human
SP-B
mRNA
(forward
5′-ATGGCTGAGTCACACCTGCTG-3′
and
reverse
5′-
TCAAAGGTCGGGGCTGTGGATAC-3′). The resulting 1,146 bp RT-PCR products were
subcloned first into the pGEM-T Easy vector (Promega) and thereafter into pcDNA5/FRT
(Invitrogen) using NotI sites. Sequencing of the two plasmid constructs (pcDNA5/FRT:SP-B/Ile
and pcDNA5/FRT:SP-B/Thr) was performed to confirm that there were no other sequence
differences between the constructs. The pcDNA5/FRT:SP-B/Ile and pcDNA5/FRT:SP-B/Thr
constructs were transfected into Flp-In-CHO cells (Invitrogen) using Fugene 6 transfection reagent
(Roche, Basel, Switzerland). Cells were cultured in nutrient mixture F-12 (Ham’s F-12; Sigma-
5
Aldrich) containing 500 μg/ml hygromycin B (Invitrogen), and SP-B–expressing clones were
selected according to the Flip-In System protocol (Invitrogen).
CHO cells were chosen for this study although the model is different from type 2 cells. Studying
type 2 cell characteristics is problematic because they are prone to fail maintaining their phenotype
in cell culture and lose their typical surfactant related features such as lamellar bodies and ability to
produce surfactant proteins; the hydrophobic SP-B and SP-C in particular. It is challenging to study
SP-B metabolism in a native context; e.g. A549 cells (which are adenocarcinomic human alveolar
basal epithelial cells) are not useful for these studies either. We envisioned that using stably
transfected cell lines, such as the Flip-In CHO cells, would bring forth certain benefits, especially
by ensuring that the integration of the cDNA into the host cell genome would be similar for both
constructs. This should reduce background variability in the secretion kinetic studies, which would
typically be present in transient transfections and also in any cell model with random DNA
integration. It has previously been shown that the initial NH2-terminal cleavage, which removes the
proSP-B peptide containing the glycosylation site of interest, is independent of cell type and occurs
soon after translation already in the ER. Therefore, it could be rationalized that in type 2 cells, any
differences of the Ile131Thr allelic variants in processing, intracellular trafficking or secretion
should originate from very early steps in the ER. Given this, although the CHO cells would
definitely not be precisely indicative of the secretion kinetics in type 2 cells, our rationale was that
if it is possible to observe any allele-specific differences even in the absence of a type 2 cellspecific regulated secretory pathway, this would clearly support the idea that this glycosylation
indeed has potential for a functional role in secretory cells in general. Being able to show functional
evidence for the glycosylation difference in the secreted protein in the cells utilizing the constant
non-regulated secretory pathway would in any case give valuable novel data and also would be
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compatible with the hypothesis that the potential allele-specific glycosylation effect on the secretion
rate would originate from very early post-translational steps.
ProSP-B detection in CHO cells and carbohydrate removal with PNGase F
Cells and culture media were assayed for SP-B/Ile and SP-B/Thr expression and secretion by
western blotting analysis. PNGase F digestion was performed to confirm the presence of
asparagine-linked oligosaccharides in the expressed proSP-B. Aliquots of CHO cell lysates and
media were denatured in 0.5% SDS and 0.4 M dithiothreitol at 100°C for 5 min, followed by
incubation with 100 U of PNGase F (New England Biolabs) and 1% Nonidet P-40 in 50 mM
sodium phosphate (pH 7.5) for 3 h at 37°C. Proteins were separated under reducing conditions
using a 12% NuPAGE Bis-Tris gels with MOPS running buffer according to the manufacturer’s
protocol (Invitrogen), and then transferred electrophoretically to an Immobilon-P polyvinylidene
difluoride (PVDF) transfer membrane (GE Healthcare, Fairfield, CT). ProSP-B was detected with
purified rabbit antiserum NFProx against SP-B Ser145–Leu160 and enhanced chemiluminescence
detection (ECL Plus western blotting detection system, GE Healthcare).
Pulse-chase labeling, immunoprecipitation, and immunoblotting
Prior to the pulse-chase labeling experiments, CHO cells stably expressing either SP-B/Ile or SPB/Thr were transferred from Ham’s F-12 to Dulbecco’s modified Eagle’s medium (DMEM, SigmaAldrich) and incubated for 1 h. Nontransfected CHO cells were used as a control for absence of SPB. After pretreatment, cells were depleted by incubating in Met-Cys–free DMEM (Sigma-Aldrich)
for 1 h. Met-Cys–free medium was replaced with fresh medium supplemented with 100 μCi/ml
[35S]methionine/cysteine (EasyTag EXPRE35S35S Protein Labeling Mix, Perkin Elmer, Waltham,
MA). After a 30 min pulse, the labeling medium was changed to DMEM supplemented with 250
mM methionine and 250 mM cysteine. Samples, both cells and media, were harvested at intervals
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of 0, 30, 60, 120, and 240 min during the 240 min chase. Cells were removed from the media by
centrifugation at 300 x g. Cell samples were washed and harvested in phosphate-buffered saline
(PBS).
Radiolabeled CHO cell pellets were lysed by sonication into lysis buffer (50 mM Tris [pH 7.4], 150
mM sodium chloride, 1% IGEPAL CA-630, and 0.5% sodium deoxycholate) containing protease
inhibitors (Complete Mini, Protease Inhibitor Cocktail Tablets, Roche). Cell debris was removed by
centrifugation. One tenth of both lysates and media was used for immunoprecipitation. Samples
were adjusted to the final immunoprecipitation buffer concentration of 50 mM Tris (pH 7.4), 150
mM sodium chloride, 5 mM EDTA, and 1% Triton X-100. Adjusted samples were precleared with
Protein A agarose (Invitrogen) and incubated for 16 h at 4°C with either nonimmune serum or antiSP-B serum (rabbit anti-human SP-B, AB3436, Chemicon). Protein A agarose beads were washed
and the bound proteins were eluted from the beads by incubation in 20 µl of NuPAGE LDS Sample
Buffer (Invitrogen) with 0.05% 2-mercaptoethanol. Samples were subjected to SDS-PAGE using
12% polyacrylamide gels and 1× MES running buffer (Invitrogen) and blotted electrophoretically
onto PVDF membrane (GE Healthcare) for autoradiography. Radioactively labeled SP-B was
detected with the PhosphorImager system (Bio-Rad). Densitometry was performed using Quantity
One software (Bio-Rad). GraphPad Prism 4 software was used to analyze the secretion kinetics.
Allelic expression imbalance (AEI) assay
Adult human lung tissue samples (n = 8) heterozygous for the SFTPB Ile131Thr polymorphism
were tested for AEI using the SNaPshot assay, which is a primer extension method based on the
addition of a single fluorescent dideoxyribonucleotide at the 3′ end of the primers designed to be
adjacent to the SNP site (ABI Prism SNaPshot Multiplex kit, Applied Biosystems, Foster City,
CA). To prepare the samples for the AEI assay, total RNA was extracted from the lung samples
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using TRI Reagent (Sigma-Aldrich). Two micrograms of total RNA was reverse transcribed to
cDNA using 200 ng/µl random hexamers (Roche), 40 U/µl RiboLock Ribonuclease Inhibitor
(Fermentas), and M-MuLV reverse transcriptase (Finnzymes, Espoo, Finland). Each cDNA was
then
amplified
in
5–7
replicate
PCR
reactions
using
forward
(5′-
GCCGATGACCTATGCCAAGAGT-3′) and reverse (5′-CCAGCAATAGGGGAGAGGAATG-3′)
primers. To remove dNTPs and primers, the PCR products were treated with rAPid Alkaline
Phosphatase (Roche) and Exonuclease I (Fermentas) before the SNaPshot reaction. The SNaPshot
primer used was 5′-GACTACTTCCAGAACCAGC-3′, and the primer extension reaction followed
the manufacturer’s protocol. Duplicates of the SNaPshot reactions, mixed with GeneScan 120 LIZ
size standard (Applied Biosystems) and formamide, were run on an ABI3130xl Genetic Analyzer
(Applied Biosystems). The results were analyzed with GeneMapper v3.7 (Applied Biosystems).
To correct for biases in the detection efficiency of different fluorophores in the SNaPshot assay,
series of different molar ratios of full-length Ile vs. Thr cDNA variant plasmids was used to
calculate a correction factor for peak ratios to be used for the assays of the lung cDNA samples.
Using this correction factor, the data was normalized for equal allelic expression to a ratio of 1.0.
Thus, deviation from the value of 1.0 should demonstrate AEI, with Thr:Ile (nucleotide peaks C:T)
ratios significantly below 1.0 indicative of preferential expression of the Ile variant mRNA or
reduced expression of the Thr variant mRNA. To prepare the internal control plasmids for the AEI
assays, we used carefully quantitated aliquots of each of the full-length SP-B cDNA plasmids SP-B
Full-Ile and Full-Thr in pGEM-T Easy (Promega), which were linearized with XhoI (New England
Biolabs) and HindIII (Pharmacia Biotech) and purified using the GeneClean Turbo (Q-BIOgene)
and GeneElute PCR Clean-Up (Sigma-Aldrich) kits. For PCR amplification and SNaPshot AEI
validation, the Ile and Thr cDNA variant plasmids were mixed at the following molar ratios: 100:0,
75:25, 50:50, 25:75, and 0:100. The 50:50 ratio was then used as a reference sample in all of the
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AEI assays. To quantitate mRNA allelic expression, the cDNA ratio of Thr and Ile allele peak
heights from the lung tissue samples were normalized with the values obtained from the reference
plasmid sample used in the same reaction. Using this procedure, a value of 1.0 represented an equal
allelic ratio; i.e., no allelic imbalance.
Statistical analyses
Statistical analyses were performed using PASW Statistics 18 Data Editor (LEAD Technologies
Inc., Charlotte, NC). All of the SFTPB polymorphisms were in Hardy–Weinberg equilibrium. To
test for a correlation between SP-B protein levels in the amniotic fluid samples (n = 251) and the
SFTPB genotypes, we used ANOVA (square root modification to achieve normally distributed
values, allowing for parametric testing) and Kruskal–Wallis (unmodified values not normally
distributed, requiring non-parametric testing) tests. Genotype-specific comparisons of the means
and the medians were performed using T-test or Mann-Whitney test, respectively. Multivariate
associations were analyzed using linear regression models with SP-B protein levels as the
dependent variable and the Ile131Thr genotype, GA and respiratory distress as independent
variables. AEI was tested using the one-sample t-test where the mean ratios of Ile and Thr variant
expressions were used as a test value against the reference value of 1.0. In statistical analyses, a p
value of <0.05 was considered significant.
Online supplement: References
1. Schutz-Geschwender A, Zhang Y, Holt T, McDermitt D, Olive DM. Quantitative, two-color
western
blot
detection
with
infrared
fluorescence.
LI-COR
Biosciences,
2004.
(http://www.licor.com/bio/PDF/IRquant.pdf.)
2. Haataja R, Ramet M, Marttila R, Hallman M. Surfactant proteins A and B as interactive genetic
determinants of neonatal respiratory distress syndrome. Hum Mol Genet 2000;9:2751-60.
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Online supplement: Figure legends
Supplemental Figure S1. Relative SP-B protein levels plotted against GA (n = 273), including 251
amniotic fluid samples from term (GA ≥ 37 weeks) and 22 samples from preterm (GA < 37 weeks)
births. The difference in SP-B levels between fetuses later diagnosed with RDS and those without
RDS was significant (p < 0.001), with lower SP-B levels in RDS group. The TTN group had
significantly lower SP-B levels than the non-TTN group (p < 0.001). Figure keys: blue diamond =
no respiratory distress, green square = TTN, yellow circle = RDS, and red triangle = pneumothorax.
The term pregnancies included in the genetic analyses are indicated with vertical line.
Supplemental Figure S2. Allele-specific mobility and N-glycosylation of human proSP-B in
transfected CHO cells: Asn129 is glycosylated in proSP-B/Thr, but not in proSP-B/Ile.
Representative gel of PNGase F-treated (+) versus untreated (−) cell lysates (A) and media (B).
ProSP-B from both Ile and Thr allele products was sensitive to PNGase F due to C-terminal
glycosylation at Asn311. However, the decrease in size was larger for the Thr allele product,
indicating that the Thr peptide contains an oligosaccharide in its NH2 terminus at Asn129, whereas
the Ile peptide does not. Smaller size of untreated proSP-B in the cell lysate than in the medium is
due to N-glycan modifications in the secretory pathway.
Supplemental Figure S3. Allelic mRNA expression ratio of Ile and Thr variant mRNAs in
heterozygous lung tissue samples (n = 8). The expression ratios of the Ile and Thr variant mRNAs
(multiple replicates), normalized by the reference sample ratio (plasmid DNA, 50:50), did not differ
from the expected value of 1.0 (p = 0.229), indicating equal mRNA expression and lack of AEI
between the two variants.
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