Previous studies have shown that pulmonary surfactant protein D (SP-D) is composed of a 43-kDa polypeptide with a short NH2-terminal domain, a collagen sequence, and a CCOH-terminal C-type lectin domain. In the present studies, ultrastructural and biochemical techniques were used to examine the quaternary structure of native rat SP-D (rSP-D). Electron microscopy of freeze-dried preparations demonstrated a highly homogeneous population of molecules with four identical rod-like arms (46 nm in length), each with an 8-9-nm diameter globular terminal expansion. The arms, which are similar in diameter to the type I collagen helix (≃4 nm), emanate from the central 'hub' in two pairs that closely parallel each other for their first 10 nm. This structure is consistent with hydrodynamic studies that predict an highly asymmetric and extended molecule (f/f0 = 3.26) with a large Stokes radius (R8 = 18 nm). Pepsin digestion gave glycosylated, trimeric collagenous fragments (43 ± 4 nm, 17 kDa/chain). Trimeric subunits containing intact triple helical domains were also liberated from SP-D dodecamers by sulfhydryl reduction under non-denaturing conditions. Digestion of rSP-D with bacterial collagenase generated a COOH-terminal carbohydrate binding fragment and a smaller peptide (≃12 kDa, unreduced) that contains interchain disulfide bonds. Electron microscopy also demonstrated higher orders of multimerization, with as many as 8 molecules associated at the hub. These studies demonstrate that SP-D is assembled as homopolymers of four identical trimeric subunits, that interactions between the amino-terminal domains of the trimers are stabilized by interchain disulfide bonds, and that SP-D molecules can associate to form complex multimolecular assemblies.
|Number of pages||9|
|Journal||Journal of Biological Chemistry|
|State||Published - Jun 24 1994|