Developmental and genetic regulation of human surfactant protein B in vivo

Aaron Hamvas, Hillary B Heins, Susan H Guttentag, Daniel J Wegner, Michelle A Trusgnich, Kate W Bennet, Ping Yang, Christopher S Carlson, Ping An, F Sessions Cole, Aaron Hamvas, Hillary B Heins, Susan H Guttentag, Daniel J Wegner, Michelle A Trusgnich, Kate W Bennet, Ping Yang, Christopher S Carlson, Ping An, F Sessions Cole

Abstract

Background: Genetic and developmental disruption of surfactant protein B (SP-B) expression causes neonatal respiratory distress syndrome (RDS).

Objectives: To assess developmental and genetic regulation of SP-B expression in vivo.

Methods: To evaluate in vivo developmental regulation of SP-B, we used immunoblotting to compare frequency of detection of mature and pro-SP-B peptides in developmentally distinct cohorts: 24 amniotic fluid samples, unfractionated tracheal aspirates from 101 infants >or=34 weeks' gestation with (75) and without (26) neonatal RDS, and 6 nonsmoking adults. To examine genetic regulation, we used univariate and logistic regression analyses to detect associations between common SP-B (SFTPB) genotypes and SP-B peptides in the neonatal RDS cohort.

Results: We found pro-SP-B peptides in 24/24 amniotic fluid samples and in 100/101 tracheal aspirates from newborn infants but none in bronchoalveolar lavage from normal adults (0/6) (p < 0.001). We detected an association (p = 0.0011) between pro-SP-B peptides (M(r) 40 and 42 kDa) and genotype of a nonsynonymous single nucleotide polymorphism at genomic position 1580 that regulates amino-terminus glycosylation.

Conclusions: Pro-SP-B peptides are more common in developmentally less mature humans. Association of genotype at genomic position 1580 with pro-SP-B peptides (M(r) 40 and 42 kDa) suggests genetic regulation of amino terminus glycosylation in vivo.

Figures

Fig. 1
Fig. 1
Western blots of mature and pro-SP-B in the unfractionated (total), large aggregate (pellet; LA), and small aggregate (supernatant; SA) surfactant fractions from simultaneously obtained trachéal aspirate (TA) and bronchoalveolar lavage (BAL) specimens from 1 older child. Mature and pro-SP-B peptides were similar in both the BAL and TA: mature SP-B was present in both SA fraction and the surface active LA fraction, while pro-SP-B was present in the SA fraction only. In Infasurf, only mature SP-B was detected, and only in the LA fraction.
Fig. 2
Fig. 2
Mature and pro-SP-B peptides from trachéal aspirate, amniotic fluid, and normal adult bronchoalveolar lavage (BAL) under nonreducing conditions. Immunoblots of TA samples from 3 newborn infants with RDS (RDS 1, 3, 4), 3 control infants (CON 1, 2, 3), 8 amniotic fluid samples (AF 1-8), 6 BAL specimens from adults (BAL 1-6), purified human SP-B (hSP-B), and Infasurf were probed with antibody to mature or pro-SP-B. Locations of common mature and pro-SP-B peptides (Mr 42, 40, 24, and 21 kDa) are indicated. TA samples from RDS 1 obtained 1 week apart (TA1 vs. TA2) demonstrate changes in mature and pro-SP-B peptide mobilities over time in the same infant. Antibody to pro-SP-B does not detect any pro-SP-B peptides in purified human SP-B or in Infasurf.
Fig. 3
Fig. 3
Mature and pro-SP-B peptides from trachéal aspirates under nonreducing and reducing conditions. Immunoblots of trachéal aspirates from 3 infants without (CON 1, 2, 3) and with (RDS 1, 2, 3) respiratory distress syndrome and purified hSP-B performed under nonreducing and reducing conditions (10% β-mercaptoethanol). Locations of common mature and pro-SP-B peptides (Mr 42, 40, 24, and 21 kDa) and reduced, mature SP-B (8 kDa) are indicated. Trachéal aspirates contain multiple mature and pro-SP-B peptides that do not resolve to an 8-kDa peptide under reducing conditions.

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