Functional characterization of the different oligomeric forms of human surfactant protein SP-D

Highlights

• rhSP-D trimers, hexamers and fuzzy balls were successfully isolated.

• These three different oligomeric forms were able to bind to bacteria.

• Only hexamers and fuzzy balls induced bacteria aggregation.

• Hexamers were isolated and found to be active structures of SP-D for the first time.

• Fuzzy balls were the most potent oligomeric form.

Abstract

Surfactant Protein D (SP-D) is a collectin protein that participates in the innate immune defense of the lungs. SP-D mediates the clearance of invading microorganisms by opsonization, aggregation or direct killing, which are lately removed by macrophages.

SP-D is found as a mixture of trimers, hexamers, dodecamers and higher order oligomers, “fuzzy balls”. However, it is unknown whether there are differences between these oligomeric forms in functions, activity or potency.

In the present work, we have obtained fractions enriched in trimers, hexamers and fuzzy balls of full-length recombinant human (rh) SP-D by size exclusion chromatography, in a sufficient amount to perform functional assays. We have evaluated the differences in protein lectin-dependent activity relative to aggregation and binding to E. coli, one of the ligands of SP-D in vivo. Fuzzy balls are the most active oligomeric form in terms of binding and aggregation of bacteria, achieving 2-fold binding higher than hexamers and 50% bacteria aggregation at very short times. Hexamers, recently described as a defined oligomeric form of the protein, have never been isolated or tested in terms of protein activity. rhSP-D hexamers efficiently bind and aggregate bacteria, achieving 50–60% aggregation at final time point and high protein concentrations. Nevertheless, trimers are not able to aggregate bacteria, although they bind to them. Therefore, SP-D potency, in functions that relay on the C-lectin activity of the protein, is proportional to the oligomeric state of the protein.

Introduction

Surfactant protein D (SP-D) is a hydrophilic protein which belongs to the collectin family [1,2]. SP-D participates in the innate immune defense of the lungs, opsonizing and promoting the clearance of invading pathogens. In addition, it participates in the regulation of the innate immune response [[3], [4], [5]]. Moreover, SP-D interacts with different receptors in inflammatory cells, regulating the release of proinflammatory mediators [2,[6], [7], [8], [9], [10], [11]]. SP-D have been also found in other mucosa in the body outside the lungs [11,12], mediating in the inflammatory response [2,13]. In addition, SP-D is implicated in the homeostasis and pool sizes of surfactant phospholipids [14,15].

SP-D monomer encloses four structural domains: the N-terminal domain, the collagen domain, the neck domain and the carbohydrate recognition domain (CRD) [4]. It has been described that SP-D is assembled in trimers – the minimal protein unit found in vivo and in vitro-, hexamers, dodecamers and large order oligomers (so called “fuzzy balls”) [1,16], with the hexamers very recently recognized as a defined oligomeric form of the protein [16]. The carbohydrate recognition domain (CRD) of SP-D, at the C-terminal end, is a key element for the majority of the protein functions. In the presence of calcium, SP-D recognizes principally carbohydrate structures in microorganisms and interact with them through the CRD [5,17].

Previous studies have attempted to isolate SP-D oligomers. In one of them, the isolation was performed using human protein (hSP-D) purified from amniotic fluid, but the resolution was limited to distinguish between low molecular weight oligomeric forms (trimers and possibly hexamers), and high molecular weight oligomers (dodecamers and fuzzy balls) [18,19]. In another study, Hartshorn and colleagues reported the isolation of recombinant human rhSP-D dodecamers and fuzzy balls, but their comparison was limited and it did not include the lower order oligomers [20].

Virus, bacteria, fungi, as well as receptors in inflammatory cells or products released by them, for example neutrophil extracellular traps (NETs), have been defined as SP-D ligands in vivo [2,4,[21], [22], [23]]. Influenza A virus (IAV) and some bacterial species, such as Escherichia coli, have been the most studied SP-D ligands in the literature [4,5]. SP-D recognizes carbohydrate structures at microbial surfaces and binds to them, inducing aggregation and opsonization of the infectious particles [11,24]. Therefore, both, bacteria and virus have been used for evaluating protein activity. The functional activity of isolated oligomeric forms of SP-D has been assessed mainly through binding assays to purified ligands, like lipopolysaccharide (LPS) or carbohydrates, IAV and bacteria. Also, virus aggregation assays have been performed. In general terms, the main conclusion obtained seem to be that large oligomers have a higher binding affinity [18], with the fuzzy balls exhibiting higher potency to aggregate IAV compared to dodecamers [20].

Despite these previous studies, it is unknown whether the different SP-D oligomeric forms could be selectively involved in certain protein functions, or whether they exhibit individually different potency to carry out specific roles. In addition, it is unknown if the SP-D hexamer has activity that is similar to the SP-D trimer or similar to higher order SP-D oligomers.

The purpose of this work has been to isolate the different oligomeric forms of recombinant human rhSP-D in order to evaluate and compare their functional activity. In particular, we pursued to achieve a higher degree of fractionation than just separating low versus high molecular weight oligomeric forms, obtaining if possible isolated trimers, hexamers, dodecamers and fuzzy balls in a sufficient amount to carry out functional assays. An additional objective was to define whether the hexamers recently found were functional structures or just an intermediate in protein oligomerization. We successfully obtained fractions highly enriched in trimers, hexamers and fuzzy balls. Fuzzy balls were the most active oligomeric form with respect to bacterial binding and aggregation assays. Hexamers triggered bacteria aggregation, although with less potency than fuzzy balls, and trimers were not sufficient to induce bacteria aggregation.