Interactions of recombinant human pulmonary surfactant protein D and SP-D multimers with influenza A

Abstract

To further study the structure and function of surfactant protein D (SP-D), recombinant human SP-D (rhSP-D) was isolated from the culture medium of Chinese hamster ovary (CHO)-K1 cells stably transfected with a full-length hSP-D cDNA. Although a significant fraction of the secreted rhSP-D was recovered as dodecamers similar to recombinant rat SP-D (rrSP-D), a major fraction accumulated as multimers of dodecamers indistinguishable from human proteinosis SP-D. As previously shown for the rat protein, rhSP-D agglutinated specific strains of influenza A virus (IAV), inhibited viral hemagglutinin activity, and protected neutrophils (PMN) from deactivation by IAV. However, the potency of rhSP-D multimers was severalfold greater than for purified dodecamers, comparable to natural, proteinosis hSP-D. Although rhSP-D multimers were also more potent than the serum collectins in mediating viral aggregation and protection of PMN, they were less potent than conglutinin in inhibiting infectivity in vitro. These studies establish that the propensity of hSP-D to form multimers of dodecamers is determined by its primary structure and demonstrate carbohydrate recognition domain valency-dependent interactions of SP-D with IAV.

Respiratory innate immune proteins differentially modulate the neutrophil respiratory burst response to influenza A virus

“…We have previously demonstrated that preincubation of IAV with dodecameric or more highly multimerized SP-D increases neutrophil uptake of the virus (13). The increased uptake was not observed with trimeric SP-D preparations (4,45).…”

“…Recombinant human SP-D (rhSP-D) was produced in CHO-K1 cells as previously described (13). For these studies the dodecameric fraction of rhSP-D was used in most instances.…”

“…For these studies the dodecameric fraction of rhSP-D was used in most instances. The dodecameric fraction and high-molecular-weight multimers fraction were purified by gel filtration as described (13).…”

“…A pool of amniotic fluid (n ϭ 6) was centrifuged at 4,000 rpm and 4°C for 30 min and purified by maltosyl agarose affinity chromatography (44). Purified SP-D was concentrated with a Vivaspin 6 (10,000 molecular weight cut off) concentrator (Vivascience, Gloucestershire, UK), and the structurally different forms of SP-D were separated by gel filtration chromatography as described (13). In brief, the samples were applied to an analytical Superose 6 column using an fast-performance liquid chromatography system (Amersham Biosciences, Freiburg, Germany) and eluted with Trisbuffered saline (TBS), pH 7.4, containing 10 mM EDTA and 0.05% emulphogen at a flow rate of 24 ml/h.…”

“…These highly multimerized forms can be separated from trimers or dodecamers by gel filtration (13). As shown in Fig.…”