Effect of hydroxylation and N187-linked glycosylation on molecular and functional properties of recombinant human surfactant protein A.
The objective of this study was to determine the effects of proline hydroxylation in the collagen-like domain and Asn(187)-linked glycosylation in the globular domain on the molecular and functional properties of human surfactant protein A1 (SP-A1). To address this issue, SP-A1 was in vitro expressed in insect and mammalian cells. Insect cells lack prolyl 4-hydroxylase activity. A glycosylation-deficient mutant SP-A1 was expressed in insect cells. In this report we present evidence that hydroxylation increased the T(m) of the collagen-like domain by 9 degrees C. Proline hydroxylation affected both the arrangement of disulfide bonding and the extent of oligomerization but did not affect conformational changes in the globular domain identified by intrinsic fluorescence. Both self-association and lipid-related functions of SP-A were clearly correlated with the thermal stability of the collagen domain and the degree of oligomerization. Structural properties and lipid-related characteristics of SP-A1 expressed in mammalian cells but not in insect cells were close to that of natural human SP-A. On the other hand, the lack of glycosylation did not affect either collagen domain stability or conformational changes induced by calcium in the globular domain. However, the lack of glycosylation favored nonspecific thermally induced aggregation of the protein.
García-Verdugo
I
I
Department of Biochemistry and Molecular Biology I, Complutense University of Madrid, 28040-Madrid, Spain.
Sánchez-Barbero
F
F
Bosch
F U
FU
Steinhilber
W
W
Casals
C
C
D015060:1,2-Dipalmitoylphosphatidylcholine / Q000737:chemistry / Q000378:metabolism; D000327:Adsorption; D000818:Animals; D016466:CHO Cells; D002118:Calcium / Q000737:chemistry / Q000494:pharmacology; D002460:Cell Line; D006224:Cricetinae; D006031:Glycosylation; D006358:Hot Temperature; D006801:Humans; D006900:Hydroxylation; D007313:Insecta; D007700:Kinetics; D008070:Lipopolysaccharides / Q000737:chemistry / Q000378:metabolism; D010743:Phospholipids / Q000737:chemistry / Q000378:metabolism; D011489:Protein Denaturation; D017434:Protein Structure, Tertiary; D037662:Pulmonary Surfactant-Associated Protein A / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D008070:Lipopolysaccharides; D010743:Phospholipids; D037662:Pulmonary Surfactant-Associated Protein A; D011994:Recombinant Proteins; D015060:1,2-Dipalmitoylphosphatidylcholine; D002118:Calcium