Soluble pattern recognition molecules: Guardians and regulators of homeostasis at airway mucosal surfaces.

Maintenance of homeostasis at body barriers that are constantly challenged by microbes, toxins and potentially bioactive (macro)molecules requires complex, highly orchestrated mechanisms of protection. Recent discoveries in respiratory research have shed light on the unprecedented role of airway epithelial cells (AEC), which, besides immune cells homing to the lung, also significantly contribute to host defence by expressing membrane-bound and soluble pattern recognition receptors (sPRR). Recent evidence suggests that distinct, evolutionary ancient, sPRR secreted by AEC might become activated by usually innocuous proteins, commonly referred to as allergens. We here provide a systematic overview on sPRR detectable in the mucus lining of AEC. Some of them become actively produced and secreted by AECs (like the pentraxins C-reactive protein and pentraxin 3; the collectins mannose binding protein and surfactant proteins A and D; H-ficolin; serum amyloid A; and the complement components C3 and C5). Others are elaborated by innate and adaptive immune cells such as monocytes/macrophages and T cells (like the pentraxins C-reactive protein and pentraxin 3; L-ficolin; serum amyloid A; and the complement components C3 and C5). Herein we discuss how sPRRs may contribute to homeostasis but sometimes also to overt disease (e.g. airway hyperreactivity and asthma) at the alveolar-air interface.

Collectins: Innate Immune Pattern Recognition Molecules.

Collectins are collagen-containing C-type (calcium-dependent) lectins which are important pathogen pattern recognising innate immune molecules. Their primary structure is characterised by an N-terminal, triple-helical collagenous region made up of Gly-X-Y repeats, an a-helical coiled-coil trimerising neck region, and a C-terminal C-type lectin or carbohydrate recognition domain (CRD). Further oligomerisation of this primary structure can give rise to more complex and multimeric structures that can be seen under electron microscope. Collectins can be found in serum as well as in a range of tissues at the mucosal surfaces. Mannanbinding lectin can activate the complement system while other members of the collectin family are extremely versatile in recognising a diverse range of pathogens via their CRDs and bring about effector functions designed at the clearance of invading pathogens. These mechanisms include opsonisation, enhancement of phagocytosis, triggering superoxidative burst and nitric oxide production. Collectins can also potentiate the adaptive immune response via antigen presenting cells such as macrophages and dendritic cells through modulation of cytokines and chemokines, thus they can act as a link between innate and adaptive immunity. This chapter describes the structure-function relationships of collectins, their diverse functions, and their interaction with viruses, bacteria, fungi and parasites.

Complement Recognition Pathways in Renal Transplantation.

The complement system, consisting of soluble and cell membrane-bound components of the innate immune system, has defined roles in the pathophysiology of renal allograft rejection. Notably, the unavoidable ischemia-reperfusion injury inherent to transplantation is mediated through the terminal complement activation products C5a and C5b-9. Furthermore, biologically active fragments C3a and C5a, produced during complement activation, can modulate both antigen presentation and T cell priming, ultimately leading to allograft rejection. Earlier work identified renal tubule cell synthesis of C3, rather than hepatic synthesis of C3, as the primary source of C3 driving these effects. Recent efforts have focused on identifying the local triggers of complement activation. Collectin-11, a soluble C-type lectin expressed in renal tissue, has been implicated as an important trigger of complement activation in renal tissue. In particular, collectin-11 has been shown to engage L-fucose at sites of ischemic stress, activating the lectin complement pathway and directing the innate immune response to the distressed renal tubule. The interface between collectin-11 and L-fucose, in both the recipient and the allograft, is an attractive target for therapies intended to curtail renal inflammation in the acute phase.

Soluble Collectin-12 (CL-12) Is a Pattern Recognition Molecule Initiating Complement Activation via the Alternative Pathway.

Soluble defense collagens including the collectins play important roles in innate immunity. Recently, a new member of the collectin family named collectin-12 (CL-12 or CL-P1) has been identified. CL-12 is highly expressed in umbilical cord vascular endothelial cells as a transmembrane receptor and may recognize certain bacteria and fungi, leading to opsonophagocytosis. However, based on its structural and functional similarities with soluble collectins, we hypothesized the existence of a fluid-phase analog of CL-12 released from cells, which may function as a soluble pattern-recognition molecule. Using recombinant CL-12 full length or CL-12 extracellular domain, we determined the occurrence of soluble CL-12 shed from in vitro cultured cells. Western blot showed that soluble recombinant CL-12 migrated with a band corresponding to ∼ 120 kDa under reducing conditions, whereas under nonreducing conditions it presented multimeric assembly forms. Immunoprecipitation and Western blot analysis of human umbilical cord plasma enabled identification of a natural soluble form of CL-12 having an electrophoretic mobility pattern close to that of shed soluble recombinant CL-12. Soluble CL-12 could recognize Aspergillus fumigatus partially through the carbohydrate-recognition domain in a Ca(2+)-independent manner. This led to activation of the alternative pathway of complement exclusively via association with properdin on A. fumigatus as validated by detection of C3b deposition and formation of the terminal complement complex. These results demonstrate the existence of CL-12 in a soluble form and indicate a novel mechanism by which the alternative pathway of complement may be triggered directly by a soluble pattern-recognition molecule.

Immunoregulatory functions of surfactant proteins.

Because the lungs function as the body's gas-exchange organ, they are inevitably exposed to air that is contaminated with pathogens, allergens and pollutants. Host-defence mechanisms within the lungs must facilitate clearance of inhaled pathogens and particles while minimizing an inflammatory response that could damage the thin, delicate gas-exchanging epithelium. Pulmonary surfactant is a complex of lipids and proteins that enhances pathogen clearance and regulates adaptive and innate immune-cell functions. In this article, I review the structure and functions of the surfactant proteins SP-A and SP-D in regulating host immune defence and in modulating inflammatory responses.

[Diagnostic significance of anti-collectin 11 in systemic lupus erythematosus].

OBJECTIVE: To analyze the role of anti-collectin 11 in the diagnosis of systemic lupus erythematosus (SLE) and in the evaluation of disease activity. METHODS: This was a cross-sectional study. Five groups of patients were enrolled: SLE active (SLE disease activity index-2000,SLEDAI-2000≥9), SLE remission (SLEDAI-2000≤4 and there was no organ involvement), rheumatoid arthritis (RA), primary Sjogren Syndrome (SS) and healthy control (HC). Serum anti-collectin 11 was detected in all the groups by ELISA. One-way ANOVA analysis and LSD-t-test as post-hoc analysis were used to compare the levels of anti-collectin 11 among all the groups. Receiver operating characteristic (ROC) curve and the area under curve (AUC) were used to analyze the value of anti-collectin 11 in the diagnosis of SLE. RESULTS: In the study, 30 patients were enrolled in each group, including 13 males and 137 females with an average age of (34±14) years (18-77 years). The age and gender of the other three groups were comparable to the two SLE groups. The difference of serum anti-collectin 11 between the SLE active group and SLE remission group was not statistically significant (88.8±16.8 vs. 89.7±24.7, P=0.896). The level of serum anti-collectin 11 was significantly higher in SLE group (as a whole) (89.1±19.4) than in RA group (49.1±22.0), SS group (56.9±30.1) and HC group (72.7±24.6) (P<0.001, P<0.001, P=0.007, respectively). The AUC was 0.806 for the diagnosis of SLE by serum anti-collectin 11. Further descriptive analysis showed that the positive rate of anti-collectin 11 was very high in the patients of SLE in whom both anti-double-stranded DNA (dsDNA) and Sm antibody were negative. The nervous system and gastrointestinal system involvement were the most common in the patients with positive anti-collectin 11. CONCLUSION: The level of serum anti-collectin 11 was significantly higher in SLE than in RA, SS and HC. anti-collectin 11 antibody had a relatively high value in the diagnosis of SLE and it might have some complementary function in the diagnosis of SLE. It might be a relatively specific autoantibody for SLE.

Collectin liver 1 and collectin kidney 1 and other complement-associated pattern recognition molecules in systemic lupus erythematosus.

The objective of this study was to explore the involvement of collectin liver 1 (CL-L1) and collectin kidney 1 (CL-K1) and other pattern recognition molecules (PRMs) of the lectin pathway of the complement system in a cross-sectional cohort of systemic lupus erythematosus (SLE) patients. Concentrations in plasma of CL-L1, CL-K1, mannan-binding lectin (MBL), M-ficolin, H-ficolin and L-ficolin were determined in 58 patients with SLE and 65 healthy controls using time-resolved immunoflourometric assays. The SLE patients' demographic, diagnostic, clinical and biochemical data and collection of plasma samples were performed prospectively during 4 months. CL-L1, CL-K1 and M-ficolin plasma concentrations were lower in SLE patients than healthy controls (P-values < 0.001, 0.033 and < 0.001, respectively). H-ficolin concentration was higher in SLE patients (P < 0.0001). CL-L1 and CL-K1 plasma concentrations in the individuals correlated in both patients and controls. Patients with low complement component 3 (C3) demonstrated a negative correlation between C3 and CL-L1 and CL-K1 (P = 0.022 and 0.031, respectively). Patients positive for anti-dsDNA antibodies had lower levels of MBL in plasma than patients negative for anti-dsDNA antibodies (P = 0.02). In a cross-sectional cohort of SLE patients, we found differences in the plasma concentrations of CL-L1, CL-K1, M-ficolin and H-ficolin compared to a group of healthy controls. Alterations in plasma concentrations of the PRMs of the lectin pathway in SLE patients and associations to key elements of the disease support the hypothesis that the lectin pathway plays a role in the pathogenesis of SLE.

Addition of glycosylation to influenza A virus hemagglutinin modulates antibody-mediated recognition of H1N1 2009 pandemic viruses.

Seasonal influenza A viruses (IAV) originate from pandemic IAV and have undergone changes in antigenic structure, including addition of glycans to the viral hemagglutinin (HA). Glycans on the head of HA promote virus survival by shielding antigenic sites, but highly glycosylated seasonal IAV are inactivated by soluble lectins of the innate immune system. In 2009, human strains of pandemic H1N1 [A(H1N1)pdm] expressed a single glycosylation site (Asn(104)) on the head of HA. Since then, variants with additional glycosylation sites have been detected, and the location of these sites has been distinct to those of recent seasonal H1N1 strains. We have compared wild-type and reverse-engineered A(H1N1)pdm IAV with differing potential glycosylation sites on HA for sensitivity to collectins and to neutralizing Abs. Addition of a glycan (Asn(136)) to A(H1N1)pdm HA was associated with resistance to neutralizing Abs but did not increase sensitivity to collectins. Moreover, variants expressing Asn(136) showed enhanced growth in A(H1N1)pdm-vaccinated mice, consistent with evasion of Ab-mediated immunity in vivo. Thus, a fine balance exists regarding the optimal pattern of HA glycosylation to facilitate evasion of Ab-mediated immunity while maintaining resistance to lectin-mediated defenses of the innate immune system.

The Lectin Pathway of Complement and Biocompatibility.

In modern health technologies the use of biomaterials in the form of stents, haemodialysis tubes, artificial implants, bypass circuits etc. is rapidly expanding. The exposure of synthetic, foreign surfaces to the blood and tissue of the host, calls for strict biocompatibility in respect to contact activation, the coagulation system and the complement system. The complement system is an important part of the initial immune response and consists of fluid phase molecules in the blood stream. Three different activation pathways can initiate the complement system, the lectin, the classical and the alternative pathway, all converging in an amplification loop of the cascade system and downstream reactions. Thus, when exposed to foreign substances complement components will be activated and lead to a powerful inflammatory response. Biosurface induced complement activation is a recognised issue that has been broadly documented. However, the specific role of lectin pathway and the pattern recognition molecules initiating the pathway has only been transiently investigated. Here we review the current data on the field.

Prokaryotic expression of ovis aries conglutinin encoding neck and carbohydrate recognition domain and its functional characterization.

Conglutinin, a soluble pattern recognition receptor of innate immune system in bovines is known for its potential defensive activity against microorganisms either by direct agglutination in the presence of calcium or by acting as opsonin. In the present study, sheep (Ovis aries) conglutinin encoding neck and carbohydrate recognition domain (rSCGN) was expressed in the E coli BL21 expression host. The recombinant conglutinin revealed molecular weight of 27 kDa in SDS PAGE and also in western blotting using antibuffalo conglutinin polyclonal serum. The protein was characterized further for its functional activity in various assays. In ELISA based sugar and LPS binding assay, the rSCGN revealed its high binding activity toward N-acetyl glucosamine and E. coli LPS in the presence and the absence of calcium ions, respectively. Hemagglutination of chicken red blood cells caused by Newcastle disease virus was not inhibited in the presence of rSCGN as it lacked complete collagenous region present in the native protein. In virus neutralization test, the recombinant protein was found to reduce multiplication of bovine herpes virus-1 propagated in MDBK cells. This prokaryotically expressed 27 kDa recombinant sheep conglutinin can serve as antigen in future studies to develop sandwich ELISA for assessing the level of native conglutinin in sheep serum.

The hybrid CL-SP-D molecule has the potential to regulate xenogeneic rejection by human neutrophils more efficiently than CD47.

OBJECTIVE: Innate immunity plays a vital role in xenotransplantation. A CD47 molecule, binding to the SIRPα expressed on monocyte/macrophage cells, can suppress cytotoxicity. Particularly, the SIRPα contains ITIM, which delivers a negative signal. Our previous study demonstrated that the binding between CL-P1 and surfactant protein-D hybrid (CL-SP-D) with SIRPα regulates macrophages' phagocytic activity. In this study, we examined the effects of human CD47 and CL-SP-D expression on the inhibition of xenograft rejection by neutrophils in swine endothelial cells (SECs). METHODS: We first examined SIRPα expression on HL-60 cells, a neutrophil-like cell line, and neutrophils isolated from peripheral blood. CD47-expressing SECs or CL-SP-D-expressing SECs were generated through plasmid transfection. Subsequently, these SECs were co-cultured with HL-60 cells or neutrophils. After co-culture, the degree of cytotoxicity was calculated using the WST-8 assay. The suppressive function of CL-SP-D on neutrophils was subsequently examined, and the results were compared with those of CD47 using naïve SECs as controls. Additionally, we assessed ROS production and neutrophil NETosis. RESULTS: In initial experiments, the expression of SIRPα on HL-60 and neutrophils was confirmed. Exposure to CL-SP-D significantly suppressed the cytotoxicity in HL-60 (p = 0.0038) and neutrophils (p = 0.00003). Furthermore, engagement with CD47 showed a suppressive effect on neutrophils obtained from peripheral blood (p = 0.0236) but not on HL-60 (p = 0.4244). The results of the ROS assays also indicated a significant downregulation of SEC by CD47 (p = 0.0077) or CL-SP-D (p = 0.0018). Additionally, the suppression of NETosis was confirmed (p = 0.0125) in neutrophils co-cultured with S/CL-SP-D. CONCLUSION: These results indicate that CL-SP-D is highly effective on neutrophils in xenogeneic rejection. Furthermore, CL-SP-D was more effective than CD47 at inhibiting neutrophil-mediated xenograft rejection.

The human cathelicidin LL-37 inhibits influenza A viruses through a mechanism distinct from that of surfactant protein D or defensins.

LL-37, the only human cathelicidin, is a cationic antimicrobial peptide with antibacterial and antifungal activity. LL-37 is released from neutrophil granules and produced by epithelial cells. It has been implicated in host defence against influenza A virus (IAV) in recent studies. We now demonstrate dose-related neutralizing activity of LL-37 against several seasonal and mouse-adapted IAV strains. The ability of LL-37 to inhibit these IAV strains resulted mainly from direct effects on the virus, since pre-incubation of virus with LL-37 was needed for optimal inhibition. LL-37 bound high-density lipoprotein (HDL), and pre-incubation of LL-37 with human serum or HDL reduced its antiviral activity. LL-37 did not inhibit viral association with epithelial cells as assessed by quantitative RT-PCR or confocal microscopy. This finding contrasted with results obtained with surfactant protein D (SP-D). Unlike collectins or human neutrophil defensins (HNPs), LL-37 did not induce viral aggregation under electron microscopy. In the electron microscopy studies, LL-37 appeared to cause disruption of viral membranes. LL-37 had additive antiviral activity when combined with other innate inhibitors like SP-D, surfactant protein A and HNPs. Unlike HNPs, LL-37 did not bind SP-D significantly. These findings indicate that LL-37 contributes to host defence against IAV through a mechanism distinct from that of SP-D and HNPs.

Diverse functions of pulmonary collectins in host defense of the lung.

Pulmonary surfactant is a mixture of lipids and proteins that covers alveolar surfaces and keeps alveoli from collapsing. Four specific proteins have been identified in surfactant. Among them, two C-type lectins, surfactant proteins A and D (SP-A and SP-D), are known to be implicated in host defense and regulation of inflammatory responses of the lung. These host defense lectins are structurally characterized by N-terminal collagen-like domains and lectin domains and are called pulmonary collectins. They prevent dissemination of infectious microbes by their biological activities including agglutination and growth inhibition. They also promote clearance of microbes by enhancing phagocytosis in macrophages. In addition, they interact with the other pattern-recognition molecules, including Toll-like receptors (TLRs) and TLR-associated molecules, CD14 and MD-2, and regulate inflammatory responses. Furthermore, recent studies have demonstrated that these collectins modulate functions of neutrophil-derived innate immune molecules by interacting with them. These findings indicate that pulmonary collectins play critical roles in host defense of the lung.

Biological functions of the novel collectins CL-L1, CL-K1, and CL-P1.

Collectins are characterized by a collagen-like sequence and a carbohydrate recognition domain and are members of the vertebrate C-type lectin superfamily. Recently, "novel collectins", different from "classical collectins" consisting of mannan-binding lectin (MBL) and surfactant proteins A and D (SP-A and SP-D), have been found by reverse genetics. These "novel collectins" consist of collectin liver 1 (CL-L1), collectin kidney 1 (CL-K1), and collectin placenta 1 (CL-P1) and are encoded by three separate genes. Experimental findings on human and animal collectins have shown that both novel collectins and classical collectins play an important role in innate immunity. Based on our recent results and those of others, in this paper, we summarize the new biological functions of these novel collectins in embryonic morphogenesis and development.

Collectin 11 (CL-11, CL-K1) is a MASP-1/3-associated plasma collectin with microbial-binding activity.

Collectins play important roles in the innate immune defense against microorganisms. Recently, a new collectin, collectin 11 (CL-11 or CL-K1), was identified via database searches. In present work, we characterize the structural and functional properties of CL-11. Under nonreducing conditions, in gel permeation chromatography recombinant CL-11 forms disulfide-linked oligomers of 100 and 200 kDa. A mAb-based ELISA estimates the concentration of CL-11 in plasma to be 2.1 µg/ml, and the presence of CL-11 in plasma was further verified by Western blotting and mass spectrometry. Mannan-binding lectin-associated serine protease 1 (MASP-1) copurified with CL-11 and the interaction in plasma with MASP-1 and/or MASP-3 was further demonstrated using ELISA. We identified the adrenal glands, the kidneys, and the liver as primary sites of expression. CL-11 lectin activity was demonstrated by ELISA and showed that CL-11 has preference for l-fucose and d-mannose. We finally show that CL-11 binds to intact bacteria, fungi, and viruses and that CL-11 decreases influenza A virus infectivity and forms complexes with DNA. On the basis of the significant concentration of CL-11 in circulation and CL-11's interaction with various microorganisms and MASP-1 and/or MASP-3, it is conceivable that CL-11 plays a role in activation of the complement system and in the defense against invading microorganisms.

Pandemic H1N1 influenza A viruses are resistant to the antiviral activities of innate immune proteins of the collectin and pentraxin superfamilies.

Acquired immune responses elicited to recent strains of seasonal H1N1 influenza viruses provide limited protection against emerging A(H1N1) pandemic viruses. Accordingly, pre-existing or rapidly induced innate immune defenses are of critical importance in limiting early infection. Respiratory secretions contain proteins of the innate immune system, including members of the collectin and pentraxin superfamilies. These mediate potent antiviral activity and act as an initial barrier to influenza infection. In this study, we have examined the sensitivity of H1N1 viruses, including pandemic virus strains, for their sensitivity to collectins (surfactant protein [SP]-D and mannose-binding lectin [MBL]) and to the pentraxin PTX3. Human SP-D and MBL inhibited virus-induced hemagglutinating activity, blocked the enzymatic activity of the viral neuraminidase, and neutralized the ability of H1N1 viruses to infect human respiratory epithelial cells in a manner that correlated with the degree of glycosylation in the globular head of the hemagglutinin. Recent seasonal H1N1 viruses expressed three to four N-glycosylation sequons on the head of hemagglutinin and were very sensitive to inhibition by SP-D or MBL, whereas A(H1N1) pandemic viruses expressed a single N-glycosylation sequon and were resistant to either collectin. Of interest, both seasonal and pandemic H1N1 viruses were resistant to PTX3. Thus, unlike recent seasonal H1N1 strains of influenza virus, A(H1N1) pandemic viruses are resistant to the antiviral activities of innate immune proteins of the collectin superfamily.

Host defence against Candida albicans and the role of pattern-recognition receptors.

Recognition of Candida albicans is mediated by several classes of pattern-recognition receptors, including Toll-like receptors and C-type lectin receptors. Cell wall components of C. albicans, interact with the pattern-recognition receptors, which are expressed by different cells, primarily antigen-presenting cells. This review aims to discuss the different pattern-recognition receptors responsible for recognition of special structures of C. albicans, which are known to activate intracellular signals that finally lead to directed and efficient host defence.

Pattern recognition receptors in the immune response against dying cells.

Pattern recognition receptors (PRR), immune sensors that discriminate self from non-self, link innate to adaptive immunity. PRR are involved in microbe internalization by phagocytes (soluble PRR and endocytic receptors) and/or cell activation (signaling PRR). PRR also recognize dying cells (i.e. modified self). Apoptotic cell recognition involves soluble bridging molecules (e.g. pentraxins) and endocytic receptors (e.g. scavenger receptors, the CD91-calreticulin complex). Apoptotic cells induce an immunosuppressive signal, avoiding the initiation of an autoimmune response. By contrast, necrotic cells, via the release of stimulatory molecules [heat shock protein (HSP), high-mobility group box 1 protein (HMGB1)], activate immune cells. This review summarizes the PRR involved in the recognition of dying cells and the consequences on the outcome of the immune response directed against dying cell antigens.

Viral aggregating and opsonizing activity in collectin trimers.

Collectins are collagenous lectins present in blood, respiratory lining fluid, and other mucosal secretions that play important roles in innate defense against infection. The collectin, surfactant protein D (SP-D), limits infection by viruses and bacteria in the respiratory tract, eye, and female genital tract. Multimeric SP-D has strong antiviral activity and is a potent viral and bacterial agglutinin and opsonin; however, trimers composed of the neck and carbohydrate recognition domain (hSP-D-NCRD) of SP-D lack these activities. We now show that, in contrast, a trimeric neck and CRD construct of bovine serum collectin CL-46 induces aggregation of influenza A virus (IAV) and potently increases IAV uptake by neutrophils. CL-46-NCRD showed calcium-dependent and sugar-sensitive binding to both neutrophils and IAV. Replacement of specific residues of the CRD of human SP-D with those found in bovine serum collectins conferred opsonizing activity. The most effective substitution involved replacement of arginine 343 with valine (hSP-D-NCRD/R343V). hSP-D-NCRD/R343V greatly increased viral uptake by neutrophils and monocytes and also potentiated neutrophil respiratory burst responses. These effects were further increased by cross-linking of hSP-D-NCRD/R343V trimers with MAbs directed against areas of the hSP-D-NCRD not involved in viral binding. Unlike the wild-type human SP-D hSP-D-NCRD, hSP-D-NCRD/R343V also induced viral aggregation. These results indicate that collectins can act as opsonins for IAV even in the absence of the collagen domain or higher order multimerization. This may involve increased affinity of individual CRDs for glycoconjugates displayed on host cells or the viral envelope.

Increasing antiviral activity of surfactant protein d trimers by introducing residues from bovine serum collectins: dissociation of mannan-binding and antiviral activity.

Collectins contribute to host defence through interactions with glycoconjugates on pathogen surfaces. We have prepared recombinant trimeric neck and carbohydrate recognition domains (NCRD) of collectins, and we now show that the NCRD of bovine conglutinin and CL-46 (like that of CL-43) have greater intrinsic antiviral activity for influenza A virus (IAV) than the human SP-D NCRD (hSP-D-NCRD). The three serum collectins differ from SP-D by having insertions adjacent to amino acid 325 and substitution of hydrophobic residues for arginine 343. We previously showed that a three amino acid (RAK) insertion, as found in CL-43, increases antiviral activity and mannan-binding activity of the hSP-D-NCRD, while the substitution of valine at 343, as in conglutinin, more strongly increased these activities. Mannan-binding activity of collectins has been considered to predict for ability to bind to high mannose glycans on viruses or other pathogens. We now show, however, that combined mutants containing the RAK insertion and R343V or R343I substitutions have greatly increased mannan-binding ability, but lower IAV binding or inhibiting activity than mutants containing R343V or R343I substitutions only. These findings indicate differences in the recognition of glycan structures of mannan and IAV by the NCRD and emphasize the importance of the flanking sequences in determining the differing interactions of human SP-D and bovine serum collectins with mannose-rich glycoconjugates on IAV and other pathogens. Of interest, we show conservation of some monoclonal antibody-binding epitopes between bovine collectin NCRD and hSP-D, suggesting shared structural motifs.