PubMed Health⌕ Search

Biomedical subjects

K B Reid

Publications and source records attributed to K B Reid.

At least 19 recordsLinked to original sources
PubMed2001

Expression and characterisation of the thrombospondin type I repeats of human properdin.

Properdin, an upregulator of the alternative complement pathway, is central to deposition of the activated complement fragment C3b on the surfaces of the pathogens, which it achieves by preventing the dissociation of the Bb catalytic subunit from the inherently labile C3bBb complexes. It is also known to bind sulphated glycoconjugates, such as sulphatides. Properdin has an unusual structure formed by oligomerisation of a rod-like monomer into cyclic dimers, trimers and tetramers. The monomer (approximately 53 kDa) contains an N-terminal region of no known homology, followed by six non-identical repeats of 60 amino acids (based on exon/intron boundaries), called 'thrombospondin type I repeats' or TSR modules. We have expressed and purified the N-terminal region and each of the individual TSR repeats in Escherichia coli. Although the individual recombinant TSRs, after a denaturation-renaturation cycle, appeared to be correctly folded modules, as judged by the one-dimensional (1D)- and 2D-nuclear magnetic resonance spectra of TSR3, they did not show binding to either C3b or sulphatide. Polyclonal antibodies were raised against each TSR and were found to be module-specific. The anti-TSR5 polyclonal antibody was found to inhibit binding of native human properdin to solid-phase C3b, or sulphatides. It could also block properdin-dependent haemolysis of rabbit erythrocytes. These results are consistent with the view that the TSR5 contains the major site in properdin which is involved in both C3b and sulphatide binding. It also suggests that a co-operative intramolecular interaction between TSRs, as found in the native molecule, is required for TSR5 to bind either C3b or sulphatides.

Amino Acid Sequence↗
PubMed2001

A recombinant homotrimer, composed of the alpha helical neck region of human surfactant protein D and C1q B chain globular domain, is an inhibitor of the classical complement pathway.

The first step in the activation of the classical complement pathway by immune complexes involves the binding of the six globular heads of C1q to the Fc regions of IgG or IgM. The globular heads of C1q (gC1q domain) are located C-terminal to the six triple-helical stalks present in the molecule, each head being composed of the C-terminal halves of one A, one B, and one C chain. The gC1q modules are also found in a variety of noncomplement proteins, such as type VIII and X collagens, precerebellin, hibernation protein, multimerin, Acrp-30, and saccular collagen. In several of these proteins, the chains containing these gC1q modules appear to form a homotrimeric structure. Here, we report expression of an in-frame fusion of a trimerizing neck region of surfactant protein D with the globular head region of C1q B chain as a fusion to Escherichia coli maltose binding protein. Following cleavage by factor Xa and removal of the maltose binding protein, the neck and globular region, designated ghB(3), formed a soluble, homotrimeric structure and could inhibit C1q-dependent hemolysis of IgG- and IgM-sensitized sheep erythrocytes. The functional properties of ghB(3) indicate that the globular regions of C1q may adopt a modular organization in which each globular head of C1q may be composed of three structurally and functionally independent domains, thus retaining multivalency in the form of a heterotrimer. The finding that ghB(3) is an inhibitor of C1q-mediated complement activation opens up the possibility of blocking activation at the first step of the classical complement pathway.

Animals↗
PubMed2001

Protective role of lung surfactant protein D in a murine model of invasive pulmonary aspergillosis.

The protective effects of intranasal administration of amphotericin B (AmB), human SP-A, SP-D and a 60-kDa fragment of SP-D (rSP-D) were examined in a murine model of invasive pulmonary aspergillosis (IPA). The untreated group of IPA mice showed no survival at 7 days postinfection. Treatment with AmB, SP-D, and rSP-D increased the survival rate to 80, 60, and 80%, respectively, suggesting that SP-D (and rSP-D) can protect immunosuppressed mice from an otherwise fatal challenge with Aspergillus fumigatus conidia.

Amphotericin B↗
PubMed2001

Surfactant proteins A and D protect mice against pulmonary hypersensitivity induced by Aspergillus fumigatus antigens and allergens.

Allergic bronchopulmonary aspergillosis (ABPA) is an allergic disorder caused by an opportunistic fungal pathogen, Aspergillus fumigatus (AFU:). Lung surfactant proteins SP-A and SP-D can interact with the glycosylated antigens and allergens of AFU:, inhibit specific IgE binding to these allergens, and block histamine release from sensitized basophils. We have now examined the therapeutic effect of exogenous administration of human SP-A, SP-D, and a recombinant fragment of SP-D (rSP-D), in a murine model of pulmonary hypersensitivity induced by AFU: antigens and allergens, which resembles human ABPA immunologically. The ABPA mice exhibited high levels of AFU:-specific IgG and IgE, blood eosinophilia, extensive infiltration of lymphocytes and eosinophils in the lung sections, and a Th2 cytokine response. Treatment with SP-A, SP-D, and rSP-D lowered blood eosinophilia, pulmonary infiltration, and specific Ab levels considerably, which persisted up to 4 days in the SP-A-treated ABPA mice, and up to 16 days in the SP-D- or rSP-D-treated ABPA mice. The levels of IL-2, IL-4, and IL-5 were decreased, while the level of IFN-gamma was raised in the splenic supernatants of the treated mice, indicating a marked shift from Th2 to Th1 response. These results clearly implicate pulmonary SP-A and SP-D in the modulation of allergic reactions.

Allergens↗
PubMed2000

Distinct pathways of mannan-binding lectin (MBL)- and C1-complex autoactivation revealed by reconstitution of MBL with recombinant MBL-associated serine protease-2.

Mannan-binding lectin (MBL) plays a pivotal role in innate immunity by activating complement after binding carbohydrate moieties on pathogenic bacteria and viruses. Structural similarities shared by MBL and C1 complexes and by the MBL- and C1q-associated serine proteases, MBL-associated serine protease (MASP)-1 and MASP-2, and C1r and C1s, respectively, have led to the expectation that the pathways of complement activation by MBL and C1 complexes are likely to be very similar. We have expressed rMASP-2 and show that, whereas C1 complex autoactivation proceeds via a two-step mechanism requiring proteolytic activation of both C1r and C1s, reconstitution with MASP-2 alone is sufficient for complement activation by MBL. The results suggest that the catalytic activities of MASP-2 split between the two proteases of the C1 complex during the course of vertebrate complement evolution.

Carrier Proteins↗
PubMed2000

Porcine lung surfactant protein D: complementary DNA cloning, chromosomal localization, and tissue distribution.

Porcine organs and lung surfactant have medically important applications in both xenotransplantation and therapy. We have started to characterize porcine lung surfactant by cloning the cDNA of porcine surfactant protein D (SP-D). SP-D and SP-A are important mediators in innate immune defense for the lung and possibly other mucosal surfaces. Porcine SP-D will also be an important reagent for use in existing porcine animal models for human lung infections. The complete cDNA sequence of porcine SP-D, including the 5' and 3' untranslated regions, was determined from two overlapping bacteriophage clones and by PCR cloning. Three unique features were revealed from the porcine sequence in comparison to SP-D from other previously characterized species, making porcine SP-D an intriguing species addition to the SP-D/collectin family. The collagen region contains an extra cysteine residue, which may have important structural consequences. The other two differences, a potential glycosylation site and an insertion of three amino acids, lie in the loop regions of the carbohydrate recognition domain, close to the carbohydrate binding region and thus may have functional implications. These variations were ruled out as polymorphisms or mutations by confirming the sequence at the genomic level in four different pig breeds. Porcine SP-D was shown to localize primarily to the lung and with less abundance to the duodenum, jejunum, and ileum. The genes for SP-D and SP-A were also shown to colocalize to a region of porcine chromosome 14 that is syntenic with the human and murine collectin loci.

Amino Acid Sequence↗
PubMed2000

C1q: structure, function, and receptors.

C1q is the first subcomponent of the C1 complex of the classical pathway of complement activation. Several functions have been assigned to C1q, which include antibody-dependent and independent immune functions, and are considered to be mediated by C1q receptors present on the effector cell surface. There remains some uncertainty about the identities of the receptors that mediate C1q functions. Some of the previously described C1q receptor molecules, such as gC1qR and cC1qR, now appear to have less of a role in C1q functions than in functions unrelated to C1q. The problem of identifying receptor proteins with complementary binding sites for C1q has been compounded by the highly charged nature of the different domains in C1q. Although newer candidate receptors like C1qR(p) and CR1 have emerged, full analysis of the C1q-C1q receptor interactions is still at an early stage. In view of the diverse functions that C1q is considered to perform, it has been speculated that several C1q-binding proteins may act in concert, as a C1q receptor complex, to bring about C1q mediated functions. Some major advances have been made in last few years. Experiments with gene targeted homozygous C1q-deficient mice have suggested a role for C1q in modulation of the humoral immune response, and also in protection against development of autoimmunity. The recently described crystal structure of Acrp-30, which is a serum protein secreted from adipocytes, has revealed a new C1q/TNF superfamily of proteins. Although the members of this superfamily may have diverse functions, there may be a common theme in their phylogeny and modular organisation of their distinctive globular domains.

Animals↗
PubMed2000

Collectins and innate immunity in the lung.

Evidence from both in vitro and in vivo studies suggests that the collectins are important elements in host innate immune defences against infectious agents. Study of the collectins in specific disease settings now raises the prospects of developing therapies exploiting these mechanisms of innate immunity.

Animals↗
PubMed2000

The roles of surfactant proteins A and D in innate immunity.

Research over the last decade on the surfactant proteins SP-A and SP-D suggests roles beyond surfactant lipid homeostasis, involving their participation in innate immune defence. SP-A and SP-D bind and agglutinate an impressive array of non-self structures, ranging from bacteria and fungi to allergens and environmental inorganic substrates. Complementing binding. SP-A and SP-D initiate and enhance immune cell ingestion and killing of targets. Recently, some exciting developments have extended and clarified their contributions to innate immunity. Knockout mice for SP-A and SP-D have been developed. The SP-A knockout confirms that SP-A plays a key role in defence against lung pathogens and reveals the underlying defense mechanisms that require SP-A. These surfactant proteins have also been shown to have important roles in modulating the immune response, instructing, yet quenching, the immune reactions in the lung. The crystal structure of SP-D plus functional studies with recombinantly altered forms of SP-A and SP-D has begun to characterise the structural motifs responsible for mediating their immune functions. Linkage and polymorphism analysis is explaining the role these genes may play in lung diseases and infection.

Binding Sites↗
PubMed2000

Fine specificity of autoantibodies to calreticulin: epitope mapping and characterization.

Extracellular calreticulin (CRT) as well as anti-CRT antibodies have been reported in patients with various autoimmune disorders and CRT has been implicated in 'epitope spreading' to other autoantigens such as the Ro/SS-A complex. In addition, antibodies against parasite forms of the endoplasmic reticulum chaperone, CRT, have been found in patients suffering from onchocerciasis and schistosomiasis. In this study, we screened sera for anti-CRT antibodies from patients with active and inactive systemic lupus ertythematosus (SLE) and primary or secondary Sjögren's syndrome. Approximately 40% of all SLE patients were positive for anti-CRT antibodies. The antigenic regions of CRT were determined using full length CRT and fragments of CRT prepared in yeast and Escherichia coli, respectively. Synthetic 15mer peptides corresponding to the major autoantigenic region of CRT (amino acids 1-289), each one overlapping by 12 amino acids, were used to map the B cell epitopes on the CRT protein recognized by autoimmune sera. Major antigenic epitopes were found to be associated with the N-terminal half of the protein in 69% of the SLE sera from active disease patients, while the C-domain was not antigenic. Major epitopes were found to be reactive with antibodies in sera from SLE patients with both active and inactive disease, spanning different regions of the N and P-domains. Sera from both healthy and disease controls and primary Sjögren's syndrome patients were non-reactive to these sequences. Limited proteolysis of CRT with two major leucocyte serine proteases, elastase and cathepsin G, demonstrated that an N-terminal region of CRT is resistant to digestion. Interestingly, some of the epitopes with the highest reactivity belong to the fragments of the protein which bind to C1q and inhibit complement activation. Whether C1q association with CRT is a pathological or protective interaction between these two proteins is currently under investigation.

Adult↗
PubMed2000

C1q receptors.

Explore the source record for details and available documents.

Amino Acid Sequence↗
PubMed2000

A novel PCR-based technique using expressed sequence tags and gene homology for murine genetic mapping: localization of the complement genes.

The complement system is a cascade of serum proteins and receptors which forms a vital arm of innate immunity and enhances the adaptive immune response. This work establishes the chromosomal localization of four key genes of the murine complement system. Mapping was performed using a novel and rapid PCR restriction length polymorphism method which was developed to exploit the murine expressed sequence tag (EST) database. This technique circumvents the laborious cDNA or genomic cloning steps of other mapping methods by relying on EST data and the prediction of exon-intron boundaries. This method can be easily applied to the genes of other systems, ranging from the interests of the individual researcher to large-scale gene localization projects. Here the complement system, probably one of the most well-characterized areas of immunology, was used as a model system. It was shown that the C3a receptor C1r and C1s genes form an unexpected complement gene cluster towards the telomeric end of chromosome 6. The second mannose binding lectin-associated serine protease gene was mapped to the telomeric end of chromosome 4, which is distinct from other complement-activating serine proteases. These results provide new insights into the evolution of this group of proteins.

Animals↗
PubMed2000

Collectin structure: a review.

Collectins are animal calcium dependent lectins that target the carbohydrate structures on invading pathogens, resulting in the agglutination and enhanced clearance of the microorganism. These proteins form trimers that may assemble into larger oligomers. Each polypeptide chain consists of four regions: a relatively short N-terminal region, a collagen like region, an alpha-helical coiled-coil, and the lectin domain. Only primary structure data are available for the N-terminal region, while the most important features of the collagen-like region can be derived from its homology with collagen. The structures of the alpha-helical coiled-coil and the lectin domain are known from crystallographic studies of mannan binding protein (MBP) and lung surfactant protein D (SP-D). Carbohydrate binding has been structurally characterized in several complexes between MBP and carbohydrate; all indicate that the major interaction between carbohydrate and collectin is the binding of two adjacent carbohydrate hydroxyl group to a collectin calcium ion. In addition, these hydroxyl groups hydrogen bond to some of the calcium amino acid ligands. While each collectin trimer contains three such carbohydrate binding sites, deviation from the overall threefold symmetry has been demonstrated for SP-D, which may influence its binding properties. The protein surface between the three binding sites is positively charged in both MBP and SP-D.

Amino Acid Sequence↗
PubMed2000

Altered airway surfactant phospholipid composition and reduced lung function in asthma.

Pulmonary surfactant in bronchoalveolar lavage fluid (BALF) and induced sputum from adults with stable asthma (n = 36) and healthy controls (n = 12) was analyzed for phospholipid and protein compositions and function. Asthmatic subjects were graded as mild, moderate, or severe. Phospholipid compositions of BALF and sputum from control subjects were similar and characteristic of surfactant. For asthmatic subjects, the proportion of dipalmitoyl phosphatidylcholine (16:0/16:0PC), the major phospholipid in surfactant, decreased in sputum (P < 0.05) but not in BALF. In BALF, mole percent 16:0/16:0PC correlated with surfactant function measured in a capillary surfactometer, and sputum mole percent 16:0/16:0PC correlated with lung function (forced expiratory volume in 1 s). Neither surfactant protein A nor total protein concentration in either BALF or sputum was altered in asthma. These results suggest altered phospholipid composition and function of airway (sputum) but not alveolar (BALF) surfactant in stable asthma. Such underlying surfactant dysfunction may predispose asthmatic subjects to further surfactant inhibition by proteins or aeroallergens in acute asthma episodes and contribute to airway closure in asthma. Consequently, administration of an appropriate therapeutic surfactant could provide clinical benefit in asthma.

Adolescent↗
PubMed1999

Cloning of gp-340, a putative opsonin receptor for lung surfactant protein D.

Surfactant protein D (SP-D) is an oligomeric C type lectin that promotes phagocytosis by binding to microbial surface carbohydrates. A 340-kDa glycoprotein (gp-340) has been shown to bind SP-D in the presence of calcium but does so independently of carbohydrate recognition. This protein exists both in a soluble form and in association with the membranes of alveolar macrophages. The primary structure of gp-340 has been established by molecular cloning, which yielded a 7,686-bp cDNA sequence encoding a polypeptide chain of 2, 413 amino acids. The domain organization features 13 scavenger receptor cysteine-rich (SRCR) domains, each separated by an SRCR-interspersed domain, except for SRCRs 4 and 5, which are contiguous. The 13 SRCR domains are followed by two C1r/C1s Uegf Bmp1 domains separated by a 14th SRCR domain and a zona pellucida domain. gp-340 seems to be an alternative spliced form of DMBT1. Reverse transcription-PCR analysis showed that the main sites of synthesis of gp-340 are lung, trachea, salivary gland, small intestine, and stomach. Immunohistochemistry revealed strong staining for gp-340 in alveolar and other tissue macrophages. Immunostaining of the macrophage membrane was either uniform or focal in a way that suggested capping, whereas other macrophages showed strong intracellular staining within the phagosome/phagolysosome compartments. In some macrophages, SP-D and gp-340 were located in the same cellular compartment. Immunoreactive gp-340 was also found in epithelial cells of the small intestine and in the ducts of salivary glands. The distribution of gp-340 in macrophages is compatible with a role as an opsonin receptor for SP-D.

Amino Acid Sequence↗
PubMed1999

Crystal structure of the trimeric alpha-helical coiled-coil and the three lectin domains of human lung surfactant protein D.

BACKGROUND: Human lung surfactant protein D (hSP-D) belongs to the collectin family of C-type lectins and participates in the innate immune surveillance against microorganisms in the lung through recognition of carbohydrate ligands present on the surface of pathogens. The involvement of this protein in innate immunity and the allergic response make it the subject of much interest. RESULTS: We have determined the crystal structure of a trimeric fragment of hSP-D at 2.3 A resolution. The structure comprises an alpha-helical coiled-coil and three carbohydrate-recognition domains (CRDs). An interesting deviation from symmetry was found in the projection of a single tyrosine sidechain into the centre of the coiled-coil; the asymmetry of this residue influences the orientation of one of the adjacent CRDs. The cleft between the three CRDs presents a large positively charged surface. CONCLUSIONS: The fold of the CRD of hSP-D is similar to that of the mannan-binding protein (MBP), but its orientation relative to the alpha-helical coiled-coil region differs somewhat to that seen in the MBP structure. The novel central packing of the tyrosine sidechain within the coiled-coil and the resulting asymmetric orientation of the CRDs has unexpected functional implications. The positively charged surface might facilitate binding to negatively charged structures, such as lipopolysaccharides.

Amino Acid Sequence↗