Structural analysis and lipid-binding properties of recombinant human surfactant protein a derived from one or both genes.
Surfactant protein A (SP-A) constitutes an important part of the innate immune defense in the lung. In humans there are two functional genes (SP-A1 and SP-A2). The functional importance of having two distinct chain types in human SP-A is undefined. Amino acid substitutions in the primary structure of the protein may have effects on structural stability or on activity. To address this issue, SP-A1, SP-A2, and coexpressed SP-A1/SP-A2 variants were in vitro expressed in insect cells, purified, and used for study. We found the following: (1) Human SP-A variants expressed in insect cells, derived from one gene (SP-A1 or SP-A2) or both genes, differ in the relative extent and heterogeneity of oligomerization. SP-A1 and SP-A2 exist in small oligomeric forms, whereas coexpressed SP-A1/SP-A2 products favor the formation of larger oligomers. (2) Circular dichroic and fluorescence spectroscopic studies identified structural differences between SP-A variants in the collagen domain, with SP-A2 being more stable than SP-A1 but not in the calcium binding region. Recombinant human SP-A variants expressed in insect cells exhibit a lower melting temperature compared to native human SP-A. Oligomerization does not increase the thermal stability of the collagen domain of coexpressed SP-A1/SP-A2. (3) The ability of SP-A to undergo self-aggregation and induce phospholipid and bacterial lipopolysaccharide aggregation is greater for SP-A2 than for coexpressed SP-A1/SP-A2, which in turn is greater than that observed for SP-A1. The presence of SP-A1 polypeptide chains in coexpressed products modulates functional capabilities of SP-A, which depend on both the collagen and globular domains.
García-Verdugo
I
I
Department of Biochemistry and Molecular Biology I, Complutense University of Madrid, 28040-Madrid, Spain.
Wang
G
G
Floros
J
J
Casals
C
C
D000483:Alleles; D000595:Amino Acid Sequence; D001665:Binding Sites; D002942:Circular Dichroism; D014644:Genetic Variation; D006801:Humans; D050356:Lipid Metabolism; D008168:Lung / Q000502:physiology; D008297:Male; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D037662:Pulmonary Surfactant-Associated Protein A / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry; D013050:Spectrometry, Fluorescence; D013816:Thermodynamics
D037662:Pulmonary Surfactant-Associated Protein A; D011994:Recombinant Proteins