Abstract
C-type lectins that contain collagen-like domains are known as collectins. These proteins are present both in the circulation and in extravascular compartments and are central players of the innate immune system, contributing to first-line defenses against viral, bacterial, and fungal pathogens. The collectins mannose-binding lectin (MBL) and surfactant protein D (SP-D) are regulated by tissue fibroblasts at extravascular sites via an endocytic mechanism governed by urokinase plasminogen activator receptor-associated protein (uPARAP or Endo180), which is also a collagen receptor. Here, we investigated the molecular mechanisms that drive the uPARAP-mediated cellular uptake of MBL and SP-D. We found that the uptake depends on residues within a protruding loop in the fibronectin type-II (FNII) domain of uPARAP that are also critical for collagen uptake. Importantly, however, we also identified FNII domain residues having an exclusive role in collectin uptake. We noted that these residues are absent in the related collagen receptor, the mannose receptor (MR or CD206), which consistently does not interact with collectins. We also show that the second C-type lectin-like domain (CTLD2) is critical for the uptake of SP-D, but not MBL, indicating an additional level of complexity in the interactions between collectins and uPARAP. Finally, we demonstrate that the same molecular mechanisms enable uPARAP to engage MBL immobilized on the surface of pathogens, thereby expanding the potential biological implications of this interaction. Our study reveals molecular details of the receptor-mediated cellular regulation of collectins and offers critical clues for future investigations into collectin biology and pathology.
Highlights
Collectins are a group of proteins that make up a central part of the innate immune system
We first utilized a urokinase plasminogen activator receptor-associated protein (uPARAP) fibronectin type-II (FNII)-domain mutant construct (Fig. 1B, U-loop-P) in which the protruding loop was replaced with the counterpart from the related PLA2R1, a receptor known to facilitate the endocytosis of soluble phospholipase A2 (PLA2) [26], but which does not interact with collagens [18]
These observations establish that the protruding loop constitutes a central element required for uPARAP to bind surfactant proteins (SP)-D and mannose-binding lectin (MBL), and that this loop is sufficient for introducing collectin-binding capacity within the framework of the FNII domain of PLA2R1
Summary
The well-conserved FNII domain is the 2nd of 10 N-terminal domains that constitute the extracellular region of uPARAP (Fig. 1A). Each of these mutants represented one of the three amino acid substitutions found when comparing the sequences of the protruding loops from the FNII domains of uPARAP and MR, respectively (Fig. 4A, residues underlined in uPARAP sequence) None of these mutations had any consequence for the collagen uptake activity (Fig. 4H), consistent with the results obtained with the U-loopMR mutant. Differences in expression levels (mAb 2h9 uptake values, Fig. 5D) for WT uPARAP and E478Q were adjusted for by normalization of uptake values recorded for the other ligands (collagen I, collagen IV, SP-D, and MBL) These experiments clearly demonstrated a role of CTLD2 in the uptake of SP-D, whereas the lack of effect toward MBL pointed to an additional layer of complexity in the structural requirements of uPARAP as a collectin receptor. This observation adds further to the notion that the interaction of uPARAP with MBL and SP-D, respectively, involves both common and distinct molecular mechanisms
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