Characterization of the Ca2+-dependent binding of annexin IV to surfactant protein A

We have shown previously that surfactant protein A (SP-A) binds to annexin IV in a Ca2+-dependent manner [Sohma, Matsushima, Watanabe, Hattori, Kuroki and Akino (1995) Biochem. J. 312, 175-181]. Annexin IV is a member of the annexin family having four consensus repeats of about 70 amino acids and a unique N-terminal tail. In the present study, the functional site of both annexin IV and SP-A for the Ca2+-dependent binding was investigated using mutant proteins. SP-A bound in a Ca2+-dependent manner to an annexin-IV truncation mutant consisting of the N-terminal domain and the first three domains (TN-1-2-3). SP-A also bound to T3-4, but this interaction was not Ca2+-dependent. SP-A bound weakly to the other truncation mutants (TN-1-2, T2-3 and T2-3-4). Each consensus repeat of annexin IV possesses a conserved acidic amino acid residue (Glu70, Asp142, Glu226 and Asp301) that putatively ligates Ca2+. Using annexin-IV DE mutants in which one, two or three residues out of the four Asp/Glu were altered to Ala by site-directed mutagenesis [Nelson and Creutz (1995) Biochemistry 34, 3121-3132], it was revealed that Ca2+ binding in the third domain is more important than in the other Ca2+-binding sites. SP-A is a member of the animal lectin group homologous with mannose-binding protein A. The substitution of Arg197 of rat SP-A with Asp or Asn eliminated binding to annexin IV, whereas the substitution of Glu195 with Gln was silent. These results suggest that the Ca2+ binding to domain 3 of annexin IV is required for the Ca2+-dependent binding by SP-A and that Arg197 of SP-A is important in this binding.

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Rat surfactant protein D enhances the production of oxygen radicals by rat alveolar macrophages

Biochemical Journal ◽

10.1042/bj2860005 ◽

1992 ◽

Vol 286 (1) ◽

pp. 5-8 ◽

Cited By ~ 81

Author(s):

J F Van Iwaarden ◽

H Shimizu ◽

P H M Van Golde ◽

D R Voelker ◽

L M G Van Golde

Keyword(s):

Alveolar Macrophages ◽

Oxygen Radicals ◽

Protein A ◽

Surfactant Protein ◽

Surfactant Protein A ◽

Surfactant Protein D ◽

Dependent Manner ◽

Concentration Dependent Manner ◽

Oxygen Radical Production ◽

Stimulation Of

Rat surfactant protein D (SP-D) was shown to enhance the production of oxygen radicals by rat alveolar macrophages. This enhancement, which was determined by a lucigenin-dependent chemiluminescence assay, was maximal after 18 min at an SP-D concentration of 0.2 micrograms/ml. Surfactant lipids did not influence the stimulation of alveolar macrophages by SP-D, whereas the oxygen-radical production of these cells induced by surfactant protein A was inhibited by the lipids in a concentration-dependent manner.

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Degradation of surfactant lipids and surfactant protein A by alveolar macrophages in vitro

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.1995.268.5.l772 ◽

1995 ◽

Vol 268 (5) ◽

pp. L772-L780 ◽

Cited By ~ 19

Author(s):

J. R. Wright ◽

D. C. Youmans

Keyword(s):

Protein A ◽

Surfactant Protein ◽

Surfactant Protein A ◽

Alveolar Type ◽

Dependent Manner ◽

Type Ii ◽

Alveolar Type Ii Cell ◽

Degradation Rates ◽

Type Ii Cell

Pulmonary surfactant is synthesized and secreted into the airspaces by the alveolar type II cell. After it is secreted, surfactant undergoes a series of poorly understood transformations resulting in formation of a surface tension-reducing surface at the air-liquid interface. The by-products of the surface film and/or other products of surfactant metabolism are eventually cleared from the alveolar space. Both the alveolar type II cell and the macrophage are thought to be involved in surfactant clearance and have been shown to internalize surfactant lipid in vitro. The goal of the current investigation was to characterize further and to quantitate the role of the macrophage in surfactant clearance by investigating the uptake and metabolism of surfactant lipids and surfactant protein A (SP-A) by macrophages in vitro. SP-A enhanced the uptake of lipids by macrophages in a time-, temperature-, and concentration-dependent manner. In contrast, neither of the collagen-like proteins SP-D or C1q enhanced the uptake. Phosphatidylcholine was rapidly degraded by macrophages and the degradation occurred both in the presence and absence of SP-A. In addition, macrophages degrade SP-A by a process that is time- and temperature-dependent. These results and calculations of uptake and degradation rates suggest that macrophages may contribute significantly to the process of surfactant clearance.

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Human surfactant protein A with two distinct oligomeric structures which exhibit different capacities to interact with alveolar type II cells

Biochemical Journal ◽

10.1042/bj3170939 ◽

1996 ◽

Vol 317 (3) ◽

pp. 939-944 ◽

Cited By ~ 16

Author(s):

Akiko HATTORI ◽

Yoshio KUROKI ◽

Hitoshi SOHMA ◽

Yoshinori OGASAWARA ◽

Toyoaki AKINO

Keyword(s):

Protein A ◽

Surfactant Protein ◽

Surfactant Protein A ◽

Alveolar Type ◽

Type Ii Cells ◽

Dependent Manner ◽

Type Ii ◽

Concentration Dependent Manner ◽

Lower Affinity ◽

Alveolar Proteinosis

The lung lavage fluids from patients with pulmonary alveolar proteinosis have been generally used as a source for human surfactant protein A (SP-A). We have recently found that a multimerized form of SP-A oligomer (alveolar proteinosis protein-I, APP-I) exists besides the normal-sized octadecamer (APP-II) in SP-As isolated from the patients. When analysed by Bio-Gel A15m column chromatography in 5 mM Tris buffer (pH 7.4), the apparent molecular masses of APP-I and APP-II were 1.65 MDa and 0.93 MDa, respectively. Gel-filtration analysis also revealed that APP-II is clearly separated from APP-I in the presence of 2 mM Ca2+ and 150 mM NaCl. We investigated the abilities of both SP-A oligomers to regulate phospholipid secretion and to bind to alveolar type II cells. Although APP-I inhibited lipid secretion, it was clearly a less effective inhibitor than APP-II. IC50 for inhibition of lipid secretion was apparently 0.23±0.08 µg/ml (0.14±0.05 nM) and 0.055±0.019 µg/ml (0.059±0.020 nM) for APP-I and APP-II, respectively. Both proteins bound to monolayers of type II cells in a concentration-dependent manner; however, APP-I clearly had a lower affinity to bind to type II cells. The apparent dissociation contants were, Kd = 2.31±0.70 µg/ml (1.40±0.43 nM) and 0.89±0.22 µg/ml (0.95±0.24 nM) for APP-I and APP-II, respectively. Excess unlabelled rat SP-A replaced 45% of 125I-APP-I and 77% of 125I-APP-II for type II cell binding. Although 125I-APP-II competed with excess unlabelled APP-I or APP-II, 125I-APP-I failed to compete and instead its binding rather increased in the presence of unlabelled APPs. The biotinylated APP-I bound to APP-I and APP-II coated on to microtitre wells in a concentration-dependent manner, indicating that APP-I interacts with APPs. This study demonstrates that the multimerized form of human SP-A oligomer exhibits the following attributes: (1) the reduced capacity to regulate phospholipid secretion from type II cells, and (2) lower affinity to bind to type II cells, and that the integrity of a flower-bouquet-like octadecameric structure of SP-A oligomer is important for the expression of full activity of this protein, indicating the importance of the oligomeric structure of mammalian lectins with collagenous domains.

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Pulmonary surfactant protein A stimulates chemotaxis of alveolar macrophage

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.1993.264.4.l338 ◽

1993 ◽

Vol 264 (4) ◽

pp. L338-L344 ◽

Cited By ~ 29

Author(s):

J. R. Wright ◽

D. C. Youmans

Keyword(s):

Pulmonary Surfactant ◽

Protein A ◽

Surfactant Protein ◽

Surfactant Protein A ◽

Macrophage Migration ◽

Dependent Manner ◽

Directed Movement ◽

Concentration Dependent Manner ◽

Pulmonary Surfactant Protein ◽

Checker Board

Pulmonary surfactant modulates several functions of alveolar macrophages including phagocytosis, killing, and chemotaxis. We hypothesized that the reported stimulatory effect of surfactant on macrophage migration was mediated by one of the surfactant proteins, SP-A. We found that macrophage migration was stimulated by SP-A in a concentration-dependent manner. A concentration of 105 micrograms SP-A/ml enhanced migration approximately 10-fold. Heat treatment or reduction and alkylation of SP-A reduced its stimulatory effect. A checker-board analysis showed that SP-A stimulated migration primarily by enhancing chemotaxis (directed movement) rather than chemokinesis (random movement). The interaction of SP-A with macrophages may be mediated at least partly by the collagen-like domain of SP-A. We speculate that SP-A may play a multifunctional role in regulating pulmonary immune response by stimulating multiple macrophage functions.

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The structure and function of surfactant protein A. Hydroxyproline- and carbohydrate-deficient mutant proteins.

Journal of Biological Chemistry ◽

10.1016/s0021-9258(17)37537-3 ◽

1994 ◽

Vol 269 (8) ◽

pp. 5833-5841

Author(s):

F.X. McCormack ◽

H.M. Calvert ◽

P.A. Watson ◽

D.L. Smith ◽

R.J. Mason ◽

...

Keyword(s):

Protein A ◽

Surfactant Protein ◽

Structure And Function ◽

Surfactant Protein A ◽

Deficient Mutant ◽

Mutant Proteins ◽

And Function

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Surfactant protein A enhances alveolar macrophage phagocytosis of a live, mucoid strain ofP. aeruginosa

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.1999.277.4.l777 ◽

1999 ◽

Vol 277 (4) ◽

pp. L777-L786 ◽

Cited By ~ 20

Author(s):

William I. Mariencheck ◽

Jordan Savov ◽

Qun Dong ◽

Michael James Tino ◽

Jo Rae Wright

Keyword(s):

Outer Membrane ◽

Fluorescent Microscopy ◽

Protein A ◽

Surfactant Protein ◽

Surfactant Protein A ◽

Dependent Manner ◽

Concentration Dependent Manner ◽

Macrophage Phagocytosis ◽

Live Bacteria ◽

Membrane Components

In this study, we investigate the interaction between surfactant protein A (SP-A) and a live, mucoid strain of Pseudomonas aeruginosa and identify a mechanism of clearance of this organism by alveolar macrophages.125I-labeled SP-A bound live, but not heat-killed, P. aeruginosaorganisms in a concentration-dependent manner. Unlabeled SP-A bound live bacteria, protein isolated from whole organisms, and specific proteins of the P. aeruginosa outer membrane. The binding of SP-A to P. aeruginosa and outer membrane components was inhibited by either EDTA or mannose. Phagocytosis assays with fluorescent microscopy demonstrated that the percentage of macrophages with internalized FITC-labeled P. aeruginosa was increased 1.8-fold (19 vs. 35%) by pretreating the live bacteria with SP-A. This finding was confirmed by direct visualization of ingested bacteria by electron microscopy. Adhering macrophages to SP-A-coated surfaces attenuated the increased uptake of P. aeruginosa pretreated with SP-A, suggesting that SP-A acts as an opsonin to stimulate macrophage phagocytosis of this strain of P. aeruginosa.

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Impact of ozone exposure on the phagocytic activity of human surfactant protein A (SP-A) and SP-A variants

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.00288.2007 ◽

2008 ◽

Vol 294 (1) ◽

pp. L121-L130 ◽

Cited By ~ 61

Author(s):

Anatoly N. Mikerov ◽

Todd M. Umstead ◽

Xiaozhuang Gan ◽

Weixiong Huang ◽

Xiaoxuan Guo ◽

...

Keyword(s):

Phagocytic Activity ◽

Protein A ◽

Surfactant Protein ◽

Redox Status ◽

Ozone Exposure ◽

Surfactant Protein A ◽

Phagocytic Index ◽

Dependent Manner

Surfactant protein A (SP-A) enhances phagocytosis of Pseudomonas aeruginosa. SP-A1 and SP-A2 encode human (h) SP-A; SP-A2 products enhance phagocytosis more than SP-A1. Oxidation can affect SP-A function. We hypothesized that in vivo and in vitro ozone-induced oxidation of SP-A (as assessed by its carbonylation level) negatively affects its function in phagocytosis (as assessed by bacteria cell association). To test this, we used P. aeruginosa, rat alveolar macrophages (AMs), hSP-As with varying levels of in vivo (natural) oxidation, and ozone-exposed SP-A2 (1A, 1A0) and SP-A1 (6A2, 6A4) variants. SP-A oxidation levels (carbonylation) were measured; AMs were incubated with bacteria in the presence of SP-A, and the phagocytic index was calculated. We found: 1) the phagocytic activity of hSP-A is reduced with increasing levels of in vivo SP-A carbonylation; 2) in vitro ozone exposure of hSP-A decreases its function in a dose-dependent manner as well as its ability to enhance phagocytosis of either gram-negative or gram-positive bacteria; 3) the activity of both SP-A1 and SP-A2 decreases in response to in vitro ozone exposure of proteins with SP-A2 being affected more than SP-A1. We conclude that both in vivo and in vitro oxidative modifications of SP-A by carbonylation reduce its ability to enhance phagocytosis of bacteria and that the activity of SP-A2 is affected more by in vitro ozone-induced oxidation. We speculate that functional differences between SP-A1 and SP-A2 exist in vivo and that the redox status of the lung microenvironment differentially affects function of SP-A1 and SP-A2.

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Characterization of the Ca2+-dependent binding of annexin IV to surfactant protein A

Biochemical Journal ◽

10.1042/0264-6021:3410203 ◽

1999 ◽

Vol 341 (1) ◽

pp. 203 ◽

Cited By ~ 8

Author(s):

Hitoshi SOHMA ◽

Carl E. CREUTZ ◽

Masaki SAITOH ◽

Hitomi SANO ◽

Yoshio KUROKI ◽

...

Keyword(s):

Protein A ◽

Surfactant Protein ◽

Surfactant Protein A ◽

Annexin Iv

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Identification and Characterization of Human Surfactant Protein A Binding Protein of Mycoplasma pneumoniae

Infection and Immunity ◽

10.1128/iai.73.5.2828-2834.2005 ◽

2005 ◽

Vol 73 (5) ◽

pp. 2828-2834 ◽

Cited By ~ 57

Author(s):

T. R. Kannan ◽

D. Provenzano ◽

J. R. Wright ◽

J. B. Baseman

Keyword(s):

Amino Acids ◽

Mycoplasma Pneumoniae ◽

Pertussis Toxin ◽

Binding Protein ◽

Polyacrylamide Gel Electrophoresis ◽

Protein A ◽

Surfactant Protein ◽

Surfactant Protein A ◽

Dependent Manner ◽

Extracellular Matrix Components

ABSTRACT Mycoplasma pneumoniae infections represent a major primary cause of human respiratory diseases, exacerbate other respiratory disorders, and are associated with extrapulmonary pathologies. Cytadherence is a critical step in mycoplasma colonization, aided by a network of mycoplasma adhesins and cytadherence accessory proteins which mediate binding to host cell receptors. Furthermore, the respiratory mucosa is enriched with extracellular matrix components, including surfactant proteins, fibronectin, and mucin, which provide additional in vivo targets for mycoplasma parasitism. In this study we describe interactions between M. pneumoniae and human surfactant protein-A (hSP-A). Initially, we found that viable M. pneumoniae cells bound to immobilized hSP-A in a dose- and calcium (Ca2+)-dependent manner. Mild trypsin treatment of intact mycoplasmas reduced binding markedly (80 to 90%) implicating a surface-associated mycoplasma protein(s). Using hSP-A-coupled Sepharose affinity chromatography and polyacrylamide gel electrophoresis, we identified a 65-kDa hSP-A binding protein of M. pneumoniae. The presence of Ca2+ enhanced binding of the 65-kDa protein to hSP-A, which was reduced by the divalent cation-chelating agent, EDTA. The 65-kDa hSP-A binding protein of M. pneumoniae was identified by sequence analysis as a novel protein (MPN372) possessing a putative S1-like subunit of pertussis toxin at the amino terminus (amino acids 1 to 226), with the remaining amino acids (227 to 591) exhibiting no homology with other subunits of pertussis toxin, other known toxins, or any reported proteins. Recombinant MPN372 (MPN372) bound to hSP-A in a dose-dependent manner, which was markedly reduced by preincubation with mouse recombinant MPN372 antisera. Also, adherence of viable M. pneumoniae cells to hSP-A was inhibited by recombinant MPN372 antisera, demonstrating that MPN372, a previously designated hypothetical protein, is surface exposed and mediates mycoplasma attachment to hSP-A.

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