Membrane Interactome of a Recombinant Fragment of Human Surfactant Protein D Reveals GRP78 as a Novel Binding Partner in PC3, a Metastatic Prostate Cancer Cell Line.

Gargi Thakur,Uday Kishore,Taruna Madan,Ravi Sirdeshmukh,Barnali Biswas,Gajanan Sathe,Indra Kundu,Susan Idicula-Thomas,Saad Alkahtani,Poonam Gautam

doi:10.3389/fimmu.2020.600660

Abstract

Surfactant protein-D (SP-D), a member of the collectin family has been shown to induce apoptosis in cancer cells. SP-D is composed of an N-terminal collagen-like domain and a calcium-dependent carbohydrate recognition domain (CRD). Recently, we reported that a recombinant fragment of human SP-D (rfhSP-D), composed of homotrimeric CRD region, induced intrinsic apoptotic pathway in prostate cancer cells. Here, we analyzed the membrane interactome of rfhSP-D in an androgen-independent prostate cancer cell line, PC3, by high resolution mass spectrometry and identified 347 proteins. Computational analysis of PPI network of this interactome in the context of prostate cancer metastasis and apoptosis revealed Glucose Regulated Protein of 78 kDa (GRP78) as an important binding partner of rfhSP-D. Docking studies suggested that rfhSP-D (CRD) bound to the substrate-binding domain of glycosylated GRP78. This was further supported by the observations that human recombinant GRP78 interfered with the binding of rfhSP-D to anti-SP-D polyclonal antibodies; GRP78 also significantly inhibited the binding of recombinant full-length human SP-D with a monoclonal antibody specific to the CRD in a dose-dependent manner. We conclude that the interaction with rfhSP-D is likely to interfere with the pro-survival signaling of GRP78.

Highlights

Surfactant protein D (SP-D) recognizes an array of carbohydrate moieties present on the microbial surfaces [1, 2]
The protein profile of the membrane as well as cytosolic fractions isolated after recombinant fragment of human SP-D (rfhSP-D) pull-down assay using rfhSP-Dtreated PC3 cells was distinct, as revealed by SDS-PAGE (Figure 2A)
Ligand blotting of the membrane and cytosolic protein fractions with rfhSP-D showed presence of rfhSP-D binding proteins in both the fractions [Figure 2B(ii)]

Summary

Introduction

Surfactant protein D (SP-D) recognizes an array of carbohydrate moieties present on the microbial surfaces [1, 2]. An important role of SP-D in allergy was noted when a recombinant fragment of human SP-D (rfhSP-D) composed of neck and CRD region induced apoptosis in activated eosinophils of allergic patients [9]; this involved p53 upregulation as demonstrated in rfhSP-D treated AML14.3D10 cells, an eosinophilic leukemic cell line [10]. Kaur et al recently reported that rfhSP-D induced apoptosis in pancreatic cancer cell lines via TNF-a/Fas pathway irrespective of the p53 status [12]. A significantly reduced expression of SP-D transcripts has been reported in lung, gastric, and breast cancers, whereas ovarian cancer tissues express more SP-D. In gastric, breast, and ovarian cancers, SP-D expression suggested a poor prognosis [14]

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Conclusion