Interaction of phospholipid liposomes with plasma membrane isolated from alveolar type II cells: effect of pulmonary surfactant protein A.

Pulmonary surfactant protein A (SP-A) augments the uptake of phospholipid liposomes containing dipalmitoylphosphatidylcholine (DPPC) by alveolar type II cells. The SP-A-mediated uptake process of lipids by type II cells have not been well understood. In the present study we investigated the SP-A-mediated interaction of phospholipids with plasma membrane isolated from alveolar type II cells. SP-A increased the amount of liposomes containing radiolabeled DPPC associated with type II cell plasma membrane by 4-fold compared to the control without SP-A when analyzed by sucrose density gradient centrifugation. This effect is dependent upon the SP-A concentration. The enhancement was inhibited by anti-SP-A antibody and EGTA. When type II cell plasma membrane and liposomes containing [14C]DPPC and [3H]triolein were coincubated with or without SP-A, analysis on sucrose density gradients revealed that the profiles of [14C]DPPC and [3H]triolein in each fraction were almost identical with or without SP-A, indicating that SP-A mediates the binding of liposomes to plasma membrane but not transfer of DPPC. SP-A increased the association of liposomes containing DPPC with the membrane by 2-fold more than that containing 1-palmitoyl-2-linoleoyl-phosphatidylcholine (PLPC). SP-A induced aggregation of phospholipid liposomes containing PLPC as well as those containing DPPC, but the final turbidity of DPPC liposomes aggregated by SP-A was only by 15% greater than that of PLPC liposomes. The amount of DPPC liposomes associated with the plasma membrane derived from type II cells was 2-fold greater than that from liver. We speculate that the SP-A-mediated interaction of lipids with type II cell plasma membrane may contribute, in part, to the lipid uptake process by type II cells.

Purification and biochemical characterization of equine pulmonary surfactant protein D.

OBJECTIVE: To characterize surfactant protein D (SP-D) isolated from bronchoalveolar lavage fluid (BALF) of healthy horses. SAMPLE POPULATION: BALF from 10 Thoroughbreds (5 males, 5 females; 26 to 40 months old) without history or clinical signs of respiratory tract disease. PROCEDURE: BALF was obtained and centrifuged at 33,000 X g. The supernatant was applied to a mannose-Sepharose 6B affinity column in the presence of calcium, and the bound protein fraction was analyzed by use of sodium dodecyl sulfate-polyacrylamide gel electrophoresis, immunoblot analysis; amino acid composition was determined and partial sequencing was done. Phospholipid binding and liposome aggregation assay were performed, using purified proteins. RESULTS: The protein isolated by use of mannose affinity matrices was SP-D. It bound carbohydrates and phosphatidylinositol, which are the characteristic features of SP-D isolated from other animal species. Amino acid analysis and partial primary sequence of the isolated protein indicated high homology with rat and human SP-D. Furthermore, immunoblot analysis indicated that equine SP-D reacted with human and rat SP-D-specific antibodies. CONCLUSION AND CLINICAL RELEVANCE: SP-D exists in equine lungs; its measurement may be useful in evaluating equine lung disease.

Purification and biochemical characterization of pulmonary surfactant protein A of horses.

OBJECTIVE: To characterize surfactant protein isolated from bronchoalveolar lavage fluids of healthy horses. ANIMALS: 10 Thoroughbreds (5 males, 5 females; 26 to 40 months old) that did not have a history or clinical signs of respiratory tract disease. PROCEDURE: Bronchoalveolar lavage fluid (BALF) was obtained and centrifuged at 33,000 X g. Lipid was removed from precipitated fractions by means of extraction with 1-butanol, and organic solvent-insoluble protein precipitates were dialyzed against Tris buffer. The suspension was centrifuged, and supernatant was placed in a mannose-Sepharose affinity column, with calcium. The bound protein fraction was analyzed by means of sodium dodecyl sulfate-polyacrylamide gel electrophoresis, western immunoblot analysis, and amino acid sequencing. A liposome-aggregation assay was also performed, using purified proteins. RESULTS: Protein isolated by use of mannose-affinity matrices was identified as surfactant protein A (SP-A). It had carbohydrate-binding and phospholipid-aggregation properties characteristic of SP-A isolated from other animal species. The partial primary sequence of the isolated protein had high homology with rat and human SP-A. Furthermore, the equine SP-A reacted with anti-human and anti-rat SP-A specific antibodies. CONCLUSION: Analysis of these findings indicated the existence of SP-A in pulmonary tissues of horses. CLINICAL RELEVANCE: Measurement of SP-A concentrations may be useful for clinicians evaluating pulmonary disease of horses.

[Primary myelofibrosis with fatal mesenteric arterial thromboembolism caused by antiphospholipid syndrome].

A 60-year-old woman was admitted to our hospital in February 1993 due to dizziness, dyspnea, abdominal pain, and high susceptibility to bleeding. Physical examination revealed livedo reticularis of the foot, but did not detect hepatosplenomegaly. Examination of the peripheral blood detected pancytopenia, leukoerythroblastosis, and tear-drop erythrocytes. Primary myelofibrosis (PMF) was diagnosed on the basis of bone marrow biopsy findings. Antiphospholipid syndrome (APS) was confirmed by positive response to anti-cardiolipin antibody and recurrent splenic infarction. Because of factor XIII deficiency, the patient experienced severe gingival bleeding after tooth extraction. Her condition was complicated by mesenteric arterial thromboembolism and she died of sepsis 5 years after onset. Although the incidence of immunopathy in PMF patients is high, few studies to date have focused on APS patients presenting with a variety of severe embolic symptoms. Our patient required careful monitoring due to bleeding tendency and thromboemboli.

[Mandibular tumor with spicule formation in multiple myeloma].

A 40-year-old male complained of swelling of the right mandible. It was a hard bone tumor which was 70 x 50 x 45 mm in size without tenderness. In serum, the level of IgG was 5814 mg/dl, IgA 95 mg/dl and IgM 8 mg/dl with monoclonal increasing of gamma and lambda-chain. No Bence Jones protein was detected. In a bone marrow aspirate atypical plasma cells were 7.3% in nucleated cells in slightly hypocellular marrow. X-ray examination disclosed a spicule formation surrounding the osteolytic focus in the mandible. In addition, a osteolytic tumor without spicule formation was found in a rib. The histological examination of biopsy specimen obtained from the mandibular tumor revealed that atypical plasma cells infiltrated the fibrous tissue with new bone formation. Spicule formation in the bone lesion of multiple myeloma is unusual.

Pulmonary surfactant protein D specifically binds to phosphatidylinositol.

Alveolar type II cells produce and secrete a complex mixture of lipids and proteins called pulmonary surfactant of which phospholipids are the major components. Surfactant proteins (SP) A, B, and C interact with phospholipids and are believed to play important roles in alveolar spaces. However, whether surfactant protein D (SP-D) interacts with phospholipids is unknown. In the present study, we examined whether SP-D binds to phospholipids and investigated phospholipid specificities of SP-D binding and the structural requirements of phospholipids for that binding using 125I-SP-D as a probe. 125I-SP-D bound exclusively to phosphatidylinositol (PI) in various phospholipids or a fraction containing phospholipids extracted from surfactant, which were developed on thin layer chromatography. 125I-SP-D also bound to PI coated on microtiter wells in a manner dependent upon the SP-D concentration. Unlabeled SP-D competed well with 125I-SP-D for PI binding and the antibody against SP-D abolished 125I-SP-D binding to PI. PI liposome also attenuated 125I-SP-D binding to the solid phase PI. Ca2+ is absolutely required for the binding of SP-D to PI. SP-D failed to bind to lyso-PI, fatty acids derived from PI digested with phospholipase A2, or diacylglycerol obtained after phospholipase C treatment of PI. SP-D bound to neither phosphatidylinositol 4-monophosphate nor phosphatidylinositol 4,5-diphosphate. We conclude that SP-D specifically binds to PI. This is the first report that demonstrates that SP-D interacts with surfactant phospholipids.

The role of the amino-terminal domain and the collagenous region in the structure and the function of rat surfactant protein D.

Surfactant protein D (SP-D) is a member of the C-type lectin superfamily with four distinct structural domains: an amino terminus involved in forming intermolecular disulfides, a collagen-like domain, a neck region, and a carbohydrate recognition domain. A collagen domain deletion mutant (CDM) of SP-D was created by site-directed mutagenesis. A second variant lacking both the amino-terminal region and the collagen-like domain was generated by collagenase treatment and purification of the collagenase-resistant fragment (CRF). The CDM expressed in CHO-K1 cells formed the covalent trimers, but not the noncovalent dodecamers, typical of native SP-D. The CRF derived from recombinant SP-D formed only monomers. The CDM bound mannose-Sepharose and phosphatidylinositol (PI) as well as SP-D, but the binding to mannosyl bovine serum albumin and glucosylceramide was diminished by approximately 60%. The CRF displayed weak binding to mannose-Sepharose and PI and essentially no binding to mannosyl bovine serum albumin and glucosylceramide. Both SP-D and CDM altered the self-aggregation of PI-containing liposomes. SP-D reduced the density and the light scattering properties of PI aggregates. These results demonstrate that the collagen-like domain is required for dodecamer but not covalent trimer formation of SP-D and plays an important, but not essential, role in the interaction of SP-D with PI and GlcCer. Removal of the amino-terminal domain of SP-D along with the collagen-like domain diminishes PI binding and effectively eliminates GlcCer binding.

Altered carbohydrate recognition specificity engineered into surfactant protein D reveals different binding mechanisms for phosphatidylinositol and glucosylceramide.

Pulmonary surfactant protein D (SP-D) is a member of the collection subgroup of the C-type lectin superfamily that binds glycosylated lipids such as phosphatidylinositol (PI) and glucosylceramide (GlcCer). We have previously reported that the carbohydrate recognition domain of SP-D plays an essential role in lipid binding. However, it is unclear how the carbohydrate binding property of SP-D contributes to the lipid binding. To clarify the relationship between the lectin property and the lipid binding activity of rat SP-D, we expressed wild-type recombinant rat SP-D (rSP-D) and a mutant form of the protein with substitutions Glu-321-->Gln and Asn-323-->Asp (SP-DE321Q,N323D) in CHO-K1 cells. The indicated mutations have previously been shown to change the carbohydrate binding specificity of surfactant protein A and mannose-binding protein from mannose > galactose to the converse. rSP-D expressed in mammalian cells was essentially identical to native rat SP-D in its lipid and carbohydrate binding properties. In contrast, SP-DE321Q,N323D was unable to bind GlcCer, but retained binding activity toward PI liposomes and solid-phase PI. The efficiency of SP-DE321Q,N323D binding to PI liposome was approximately 50% of that of rSP-D in the presence of 5 mM Ca2+, but equivalent at 20 mM Ca2+. Carbohydrates competed for SP-D binding to PI such that maltose > galactose for rSP-D, and the order was reversed for SP-DE321Q,N323D. Furthermore, SP-DE321Q,N323D could bind to digalactosyldiacylglycerol more effectively than rSP-D. These results suggest the following. 1) The carbohydrate binding specificity of SP-DE321Q,N323D was changed from a mannose-glucose type to a galactose type; 2) the GlcCer binding property of SP-D is closely related to its sugar binding activity; and 3) the PI binding activity is not completely dependent on its carbohydrate binding specificity.

[A supernumerary primary tooth inducing impaction of primary incisor: a case report].

A case of a primary supernumerary tooth in a 18 month old girl was reported. 1. The supernumerary tooth was located in the maxillary incisor area. 2. The maxillary left primary central incisor and the primary supernumerary tooth were impacted. 3. The clinical and radiographic findings indicated that the primary tooth in the mesial position was the supernumerary tooth, and therefore it was removed. 4. Three days after extraction of the mesiodens, the left primary central incisor erupted. The tooth completely erupted in two months. 5. The radiographic examination showed no supernumerary succedenous teeth.

Epitope mapping for monoclonal antibodies identifies functional domains of pulmonary surfactant protein A that interact with lipids.

Pulmonary surfactant protein A (SP-A) contains 4 domains: a disulfide forming amino terminus, a collagen-like region, a neck region, and a carbohydrate recognition region. The protein binds the lipids dipalmitoylphosphatidylcholine and galactosylceramide and induces aggregation of phospholipid vesicles. SP-A also inhibits lipid secretion and enhances the uptake of phospholipid by alveolar type II cells. Previously described monoclonal antibody 1D6 blocks the inhibitory effect of SP-A on lipid secretion by type II cells, but antibody 6E3 has no effect. In the present study we mapped the epitopes for monoclonal antibodies 1D6 and 6E3 by enzyme-linked immunoassay of recombinant proteins expressed using the baculovirus system, and investigated the domain that is responsible for the SP-A interactions with lipid. Monoclonal antibody 1D6 bound to mutant SP-As in which the neck portion of the molecule was deleted or substituted with that of mannose-binding protein A, but 6E3 failed to bind to these mutants. In contrast, 1D6 did not bind to a chimera in which the carbohydrate recognition domain (CRD) was substituted with that of surfactant protein D (SP-D). In addition, 1D6 failed to recognize antigen in cells infected with the recombinant virus directing the synthesis of a Cys204-Cys218 (small disulfide loop) deletion within the CRD. Antibody 1D6 completely blocked the binding of SP-A to dipalmitoylphosphatidylcholine and galactosylceramide and liposome aggregation. By comparison, 6E3 failed to completely attenuate the interactions of SP-A with lipids. However, both 6E3 and 1D6 blocked the uptake of lipid by type II cells that is caused by SP-A. From these data, we conclude that: 1) the epitope for antibody 6E3 is located at the neck domain of SP-A and that for antibody 1D6 is at the small loop region in the CRD; 2) the CRD is essential for the SP-A functions of lipid binding, liposome aggregation, the inhibitory effect on lipid secretion, and the augmentation of lipid uptake by type II cells, and these activities are largely attributable to amino acid residues within the steric inhibitory footprint of 1D6 bound to the small disulfide loop region; and 3) the neck domain of SP-A may also be involved in the process of SP-A-mediated uptake of phospholipids by alveolar type II cells.

Chimeras of surfactant proteins A and D identify the carbohydrate recognition domains as essential for phospholipid interaction.

Pulmonary surfactant proteins A (SP-A) and D (SP-D) possess similar structure as members of the mammalian C-type lectin superfamily. Both proteins are composed of four characteristic domains which are: 1) an NH2-terminal domain involved in interchain disulfide formation (denoted A1 domain for SP-A or D1 for SP-D); 2) a collagenous domain (denoted A2 or D2); 3) a neck domain (denoted A3 or D3); and 4) a carbohydrate recognition domain (denoted A4 or D4). SP-A specifically binds to dipalmitoylphosphatidylcholine, the major lipid component of surfactant, and can regulate the secretion and recycling of this lipid by alveolar type II cells. SP-D binds to phosphatidylinositol (PI) and glucosylceramide (GlcCer), and its role in alveolar lipid metabolism remains to be clarified. To understand the relationship between the structure and the function of both proteins with respect to their interaction with lipids, we expressed recombinant wild type rat SP-D (rSP-D) and chimeric molecules of SP-A and SP-D (A1A2A3D4, A1A2D3D4, and D1D2A3A4) using a baculovirus expression system, and performed lipid binding and aggregation assays. The rSP-D effectively competed with 125I-labeled native rat SP-D in a solid phase binding assay to PI and GlcCer in a manner nearly identical to native SP-D. The rSP-D also bound to PI liposomes with approximately half the affinity of native rat SP-D. Chimera A1A2D3D4 competed with iodinated SP-D in the solid phase binding assay to both PI and GlcCer. This chimera did not bind to dipalmitoylphosphatidylcholine (DPPC) liposomes or induce their aggregation. Chimera A1A2A3D4 did not bind solid phase PI or GlcCer but was equivalent to rSP-D in binding to PI liposomes. This chimera exhibited weak binding to DPPC but failed to aggregate DPPC liposomes. Chimera D1D2A3A4 failed to bind PI and GlcCer and bound weakly to DPPC liposomes but was quite effective at inducing aggregation of DPPC liposomes. These findings demonstrate that the D3 plus D4 domains of SP-D play a role in lipid binding and that the D4 domain is essential for PI binding. Furthermore, the A3 domain of SP-A cannot account for all the lipid binding activity of this protein. In addition, the results implicate the A4 domain of SP-A as an important structural domain in lipid aggregation phenomena.

Preparation of silica gel-bonded amylose through enzyme-catalyzed polymerization and chiral recognition ability of its phenylcarbamate derivative in HPLC.

Amylose was prepared by enzymatic polymerization of alpha-D-glucose 1-phosphate dipotassium catalyzed by a phosphorylase using two kinds of the primers derived from maltopentaose, and then it was chemically bonded to silica gel to be used as a chiral stationary phase (CSP) in high-performance liquid chromatography. In method I, maltopentaose was first lactonized and allowed to react with (3-aminopropyl)triethoxysilane to form an amide bond. Amylose chains with a desired chain length and a narrow molecular weight distribution were then constructed by the enzymatic polymerization. The resulting amylose bearing a trialkoxysilyl group at the terminal was allowed to react with silica gel for immobilization. In method II, maltopentaose was first oxidized to form a potassium gluconate at the reducing terminal. After the enzymatic polymerization was performed with the potassium gluconate, the amylose end was lactonized to be immobilized to 3-aminopropyl-silanized silica gel through amide bond formation. Two amylose-conjugated silica gels thus obtained were treated with a large excess of 3,5-dimethylphenyl isocyanate to convert hydroxy groups of amylose to corresponding carbamate residues. The CSP derived through method II was superior in chiral recognition to the CSP derived from method I and showed better resolving power and higher durability against solvents such as tetrahydrofuran compared with a coated-type CSP. Influences of degree of polymerization of amylose, the spacer length between amylose and silica gel, and mobile phase compositions on chiral recognition were investigated.

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

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 NaCI. 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 microgram/ml (0.14 +/- 0.05 nM) and 0.055 +/- 0.019 microgram/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, K(d) = 2.31 +/- 0.70 microgram/ml (1.40 +/- 0.43 nM) and 0.89 +/- 0.22 microgram/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.