Purification of Native Surfactant Protein SP-A from Pooled Amniotic Fluid and Bronchoalveolar Lavage (original) (raw)
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Purification of Surfactant Protein D (SP-D) from Pooled Amniotic Fluid and Bronchoalveolar Lavage
Methods in Molecular Biology, 2013
Surfactant protein SP-D is a multimeric collagenous lectin, called collectin. SP-D is a multifunctional, pattern recognition innate immune molecule, which binds in a calcium dependent manner to an array of carbohydrates and lipids, thus offering resistance to invading pathogens, allergen challenge, and pulmonary infl ammation. SP-D is predominantly found in the endoplasmic reticulum of type 2 pneumocytes and in the secretory granules of Clara or non-ciliated bronchiolar cells. The highest expression of SP-D is observed in the distal airways and alveoli. There is also an extra pulmonary existence of SP-D. The common sources of native full-length human SP-D are bronchoalveolar lavage (BAL) washings from normal or preferably patients suffering from alveolar proteinosis who overproduce SP-D in the lungs. Amniotic fl uid collected at the term during parturition is another reasonable source. Here, we describe a simple and rapid method of purifying native SP-D away from SPA which is also present in the same source. We also describe procedures of expressing and purifying a recombinant fragment of human SP-D (rhSP-D) comprising trimeric neck and carbohydrate recognition domains that has been shown to have therapeutic effects in murine models of allergy and infection.
Isolation of human surfactant protein A from amniotic fluid
The Malaysian journal of pathology
Surfactant protein A (SP-A) is one of the four known surfactant-associated proteins found in human lungs. It plays a major role in determining regulation of surfactant uptake and resecretion. Qualitative and quantitative deficiencies of SPA may contribute to neonatal respiratory distress syndrome. The measurement of its level in amniotic fluid or neonatal tracheal aspirate may be useful in the assessment of replacement therapy using natural or synthetic surfactants. In order to develop an in-house immunoassay to detect the level of SPA , we used a discontinuous sucrose density gradient to isolate SPA from amniotic fluid. Polyacrylamide gel electrophoresis was carried out on the isolates with low molecular weight markers. We successfully isolated SPA from 12 out of 31 samples of amniotic fluid. The isolates were found to be relatively pure and have a molecular weight of about 35 kD. The isolated SPA were used as immunogens to raise antibodies in rabbits for the immunoassay .
Expression and Localization of Lung Surfactant Protein A in Human Tissues
American Journal of Respiratory Cell and Molecular Biology, 2003
Lung surfactant protein A (SP-A) is a collectin produced by alveolar type II cells and Clara cells. It binds to carbohydrate structures on microorganisms, initiating effector mechanisms of innate immunity and modulating the inflammatory response in the lung. Reverse transcriptase-polymerase chain reaction was performed on a panel of RNAs from human tissues for SPA mRNA expression. The lung was the main site of synthesis, but transcripts were readily amplified from the trachea, prostate, pancreas, and thymus. Weak expression was observed in the colon and salivary gland. SPA sequences derived from lung and thymus mRNA revealed the presence of both SP-A1 and SP-A2, whereas only SP-A2 expression was found in the trachea and prostate. Monoclonal antibodies were raised against SPA and characterized. One of these (HYB 238-4) reacted in Western blotting with both reduced and unreduced SPA , with N-deglycosylated and collagenase-treated SPA , and with both recombinant SP-A1 and SP-A2. This antibody was used to demonstrate SPA in immunohistochemistry of human tissues. Strong SPA immunoreactivity was seen in alveolar type-II cells, Clara cells, and on and within alveolar macrophages, but no extrapulmonary SPA immunoreactivity was observed. In contrast to lung surfactant protein D (SP-D), which is generally expressed on mucosal surfaces, SPA seems to be restricted to the respiratory system. Lung surfactant protein A (SP-A) is a member of the collectin family, a group of oligomeric proteins in which a collagenous region is connected by an ␣-helical neck region to a C-type lectin or carbohydrate-recognition domain (CRD) (1-3). The structural subunit consists of three such polypeptide chains, and the functional protein is made up of six subunits linked by interchain disulfide bridges near the N-terminus (4). Post-translational modifications include complex N-linked glycosylation at Asn 187 in the CRD (4). Two genes encoding SPA transcripts, SP-A1 and SP-A2, are found in humans (2, 3, 5), and several alleles are known for each gene (6). Transcripts of both genes undergo
Journal of Immunological Methods, 1998
A simple procedure has been developed for the purification of the surfactant proteins SP-A and SP-D from lung lavage of patients with alveolar proteinosis. The SP-D is purified by fractionation of the supernatant obtained after spinning the lavage at 10 000 = g for 40 min, while the bulk of the SP-A is purified by fractionation of the pellet. The supernatant is applied to a maltosyl-agarose column and the bound SP-D is specifically eluted using MnCl . The pellet is solubilised in 6 M urea and, 2 following renaturation, the solubilised proteins are applied to maltosyl-agarose and SP-A eluted using a gradient of EDTA. Both SP-A and SP-D are further purified by gel-filtration on Superose-6. This procedure has also been used to prepare successfully human SP-A and SP-D from amniotic fluid and may be generally applicable to the isolation of these surfactant proteins from lung washings obtained from other species. q
Surfactant Protein (SP)-A and SP-D as Antimicrobial and Immunotherapeutic Agents
Recent Patents on Anti-Infective Drug Discovery, 2010
Surfactant protein (SP)-A and SP-D belong to the "Soluble C-type Lectin" family of proteins and are collectively known as "Collectins". Based on their ability to recognize pathogens and to regulate the host defense, SPA and SP-D have been recently categorized as "Secretory Pathogen Recognition Receptors". SPA and SP-D were first identified in the lung; the expression of SPA and SP-D has also been observed at other mucosal surfaces, such as lacrimal glands, gastrointestinal mucosa, genitourinary epithelium and periodontal surfaces. Since the role of these proteins is not fully elucidated at other mucosal surfaces, the focus of this article is on lung-SPA and SP-D. It has become clear from research studies performed over a number of years that SPA and SP-D are critical for the maintenance of lung homeostasis and the regulation of host defense and inflammation. However, none of the surfactant preparations available for clinical use have SPA or SP-D. A review is presented here on SPA and SP-D-deficiencies in lung diseases, the importance of the administration of SPA and SP-D, and recent patents and research directions that may lead to the design of novel SPA or SP-D-based therapeutics and surfactants.
Surfactant proteins and the inflammatory and immune response in the lung
2009
Surfactant proteins are important for regulating surfactant activity and innate host defence; in particular, polymorphisms in intron 4 of the SP-B gene and dominant mutations of SP-C have been associated with bronchopulmonary dysplasia. The innate immune system is older and consists of soluble proteins, which bind microbial products and phagocytic leukocytes resembling primitive amebae, which float through the bloodstream and migrate into tissues at sites of inflammation, or reside in tissue waiting for foreign material. The innate immune system is always active and is immediately responsive, ready to recognize and inactivate microbial products entering lungs and other tissues. Pro-inflammatory cytokines (interleukins IL-1β, IL-6 and soluble ICAM-1) are present in lung lavage fluid from day 1 in premature infants with respiratory distress and reach a peak in the second week. IL-1β induces the release of inflammatory mediators, activating inflammatory cells and up-regulating adhesion...
Archives of Disease in Childhood, 2003
By lowering surface tension at the air-water interface in the surfactant deficient premature lung, exogenous surfactant replacement therapy for neonatal respiratory distress syndrome has been highly successful in decreasing mortality after preterm birth. It has emerged in recent years that surfactant components not present in current surfactant formulations-particularly surfactant associated proteins A and D (SP-A and SP-D)-have additional roles in host defence distinct from the surface tension lowering effects of surfactant. SPA and SP-D are calcium dependent carbohydrate binding proteins of the innate immune system important in the first line defence of the lung against microorganisms and in the control of lung inflammation. This review addresses the possibility that recently developed recombinant forms of SP-D could be useful therapeutically in attenuating inflammatory processes in neonatal chronic lung disease, cystic fibrosis, and emphysema.
Surfactant proteins SP-A and SP-D in human health and disease
Archivum immunologiae et therapiae experimentalis
Surfactant proteins A (SP-A) and D (SP-D) are lung surfactant-associated hydrophilic proteins that have been implicated in surfactant homeostasis and pulmonary innate immunity. They are collagen-containing C-type (calcium-dependent) lectins, called collectins, and are structurally similar to mannose-binding protein of the lectin pathway of the complement system. Being carbohydrate pattern-recognition molecules, they recognize a broad spectrum of pathogens and allergens via the lectin domain, with subsequent activation of immune cells via the collagen region, thus offering protection against infection and allergenic challenge. SP-A and SP-D have been shown to be involved in viral neutralization, clearance of bacteria, fungi, and apoptotic and necrotic cells, down-regulation of allergic reaction, and resolution of inflammation. Studies on single-nucleotide polymorphism, protein levels in broncho-alveolar lavage, and gene knock-out mice have clearly indicated an association between SP-...