E. Holm Nielsen

Serum Amyloid P Component Binds to Influenza A Virus Haemagglutinin and Inhibits the Virus Infection In Vitro

Serum amyloid P component (SAP) is a member of the phylogenetically conserved and structurally related group of proteins called pentraxins. SAP exhibits multispecific calcium‐dependent binding to oligosaccharides with terminal N‐acetyl‐galactosamine, mannose and glucuronic acid. The authors report that SAP can bind to influenza A virus and inhibit agglutination of erythrocytes mediated by the virus subtypes H1N1, H2N2 and H3N2. SAP also inhibits the production of haemagglutinin (HA) and the cytopathogenic effect of influenza A virus in MDCK cells. The binding of SAP to the virus requires physiological calcium concentrations and is blocked by specific SAP antibodies. Denaturated and renaturated SAP retained inhibition of HA. Electron microscopy shows Ca2+‐dependent binding of SAP to spikes on the viral envelope and immunoblotting indicates that SAP binds to a 50–55 kDa peptide corresponding to the mass of the HA1 peptide. Of several monosaccharides tested only D‐mannose interfered with SAPs inhibition of both HA and infectivity. The glycosaminoglycans heparan sulfate and heparin, which bind SAP, reduced SAPs binding to the virus. The results indicate that the inhibition by SAP is due to steric effects when SAP binds to terminal mannose on oligosaccharides localized close to the sialic acid‐binding site of the HA trimer.

Purification, Subunit Characterization and Ultrastructure of Three Soluble Bovine Lectins: Conglutinin, Mannose-Binding Protein and the Pentraxin Serum Amyloid P-Component

Conglutinin and mannose‐binding protein (MBP) are members of the C‐type lectins which are widely present in mammalian plasma. Serum amyloid P‐component (SAP) is a member of the pentraxin family with lectin properties. A scheme for the partial purification of all three lectins by carbohydrate affinity chromatography and selective elution was developed. The purification was monitored by SDS‐PAGE, Western blotting and electron microscopy. Binding of the lectins to Sephadex‐iC3b. their collagenase sensitivity, and the size and antibody reactivity of their subunits was investigated. The demonstration, by SDS‐PAGE, of 25‐kDa subunits, which were unaffected by collagenase treatment but bound to Sephadex‐iC3b and antibodies to human SAP, indicated the existence of bovine SAP. Bovine conglutinin (BK) also showed calcium‐dependent binding to Sephadex‐iC3b, whereas bovine MBP did not. The binding of BK was inhibitable with GlcNAc. A 3000‐fold increase in BK activity (ELISA) was obtained in eluates from Sephadex iC3b. SDS‐PAGE analyses of BK and MBP revealed subunits with an Mr of 43 kDa and 30 kDa, respectively. These subunits were sensitive to collagenase treatment which reduced the Mr to 20 kDa. Electron micrographs revealed a prominent flexible tertramer molecule (diameter 96 nm) in the BK preparations, a predominantly hexameric structure (diameter 30 nm) in the MBP preparations, and single annular pentameric disc‐like molecules (diameter 11 nm) in the SAP preparations.

Isolation and Characterization of Porcine Mannan-Binding Proteins of Different Size and Ultrastructure

The authors report on the purification and characterization of mannan‐binding proteins (MBP) isolated from porcine serum. The MBPs were purified by use of PEG precipitation, affinity chromatography on mannan‐Sepharose, protein A‐ and anti‐porcine IgM‐Sepharose followed by gel filtration. The MBP proteins were collagenase sensitive and showed γ1‐γ2‐electrophoretic mobility. The MBP designated pMBP‐28 had a molecular mass of 28 kDa when analysed on SDS‐PAGE under reducing conditions and eluted corresponding to a molecular mass of approximately 700 kDa on gel filtration chromatography. Electron micrographs of pMBP‐28 revealed an oligomeric protein similar to rodent MBP‐A and human MBP but with a predominance of penta‐ and hexameric molecules. Another protein designated pMBP‐27 was composed of peptides of 27 kDa and had an Mr of 300–350 kDa on gel filtration chromatography. Electron microscopy of pMBP‐27 showed dimer and trimer molecules; the trimers without distinct stalk regions. The N‐terminal 26 (pMBP‐27) and 24 (MBP‐28) amino acid residues showed 54% and 58% identity with human MBP. pMBP‐28 showed a higher degree of sequence similarity to rat and mouse MBP‐A (60% identity) than to mouse and rat MBP‐C (41–45% identity). Both pMBPs exhibited Ca2+‐dependent binding to D‐mannose immobilized on agarose but no significant binding to N‐acetyl‐D‐glucosamine‐ or fucose‐agarose. The results further suggested the presence of a third pMBP which copurified with pMBP‐27 but this protein was not sequenced.

MANNAN-BINDING PROTEIN FORMS COMPLEXES WITH ALPHA -2-MACROGLOBULIN. A PROPOSED MODEL FOR THE INTERACTION

We report that α‐2‐macroglobulin (α2M) can form complexes with a high molecular weight porcine mannan‐binding protein (pMBP‐28). The α2M/pMBP‐28 complexes were isolated by PEG‐precipitation and affinity chromatography on mannan‐Sepharose, protein A‐Sepharose and anti‐IgM Sepharose. The occurrence of α2M/pMBP‐28 complexes was further indicated by crossed immunoelectrophoresis and by use of an anti‐α2M affinity column and chelating Sepharose loaded with Zn2+. The eluates from these affinity columns showed α2M subunits (94 and 180 kDa) and pMBP subunits (28 kDa) in SDS‐PAGE, which reacted with antibodies against α2M and pMBP‐28, respectively, in Western blotting. Furthermore, the α2M/pMBP‐28 complexes were demonstrated by electron microscopy, Fractionation of pMBP‐containing D‐mannose eluate from mannan‐Sepharose on Superose 6 showed two protein peaks which reacted with anti‐C1 s antibodies in ELISA, one of about 650–800 kDa, which in addition contained pMBP‐28 and anti‐α2M reactive material, the other with an Mr of 100–150 kDa. The latter peak revealed rhomboid molecules (7 × 15 nm) in the electron microscope and a 67 kDa band in SDS‐PAGE under reducing conditions. This band was also seen in eluates from the anti‐α2M and chelating Sepharose columns. Based on these observations and previous findings by other investigators of a serine protease with about 67 kDa subunits which copurifies with human MBP we propose a model for the interaction of pMBP‐28 with α2M.

Serum amyloid P component inhibits influenza A virus infections: in vitro and in vivo studies

Serum amyloid P component (SAP) binds in vitro Ca(2+)-dependently to several ligands including oligosaccharides with terminal mannose and galactose. We have earlier reported that SAP binds to human influenza A virus strains, inhibiting hemagglutinin (HA) activity and virus infectivity in vitro. These studies were extended to comprise five mouse-adapted influenza A strains, two swine influenza A strains, a mink influenza A virus, a ferret influenza A reassortant virus, a influenza B virus and a parainfluenza 3 virus. The HA activity of all these viruses was inhibited by SAP. Western blotting showed that SAP bound to HA trimers, monomers and HA1 and HA2 subunits of influenza A virus. Binding studies indicated that galactose, mannose and fucose moieties contributed to the SAP reacting site(s). Intranasal administration of human SAP to mice induced no demonstrable toxic reactions, and circulating antibodies against SAP were not detected. Preincubation of virus (A/Japan/57) with SAP prevented primary infection of mice and development of antiviral antibodies. After a single intranasal administration of SAP (40 microg) 1 h before primary infection with virus (2LD(50)), nine out of 10 mice survived on day 10 and these mice approached normal body weight, whereas control mice (one out of five surviving on day 10) died. The data provide evidence of the potential of intranasally administered SAP for prophylactic treatment of influenza A virus infections in humans.

Native Human Serum Amyloid P Component is a Single Pentamer

Serum amyloid P component (SAP) and C‐reactive protein (CRP) are members of the pentraxin protein family. SAP is the precursor protein to amyloid P component present in all forms of amyloidosis. The prevailing notion is that SAP in circulation has the form of a double pentameric molecule (decamer) whereas CRP is a single pentameric molecule.

Ca2+-Mg2+-activated adenosine triphosphatase in plasma and granule membranes in non-secreting and secreting mast cells

Abstract An adenosine triphosphatase (ATP) activated by Ca2+ or Mg2+ is shown morphologically on the outer surface of non-secreting and secreting rat peritoneal mast cells. ATPase having the same properties is also seen on the external surface of the other peritoneal cells, i.e. macrophages, mononuclear cells and lymphocytes. When histamine release from the mast cells was induced by exposing them to antigen (anaphylactic reaction) or compound 48/80, ATPase activated by Ca2+ or Mg2+ could in addition be demonstrated in the granule membranes. Granule membrane ATPase is also shown in non-secreting mast cells after freezing and thawing. ATPase on the outer surface of the plasma membrane is seen in the secreting mast cells as in the non-secreting cells except in the areas where the plasma membrane fuses with the granule membrane. The role of ATPase in granule secretion process has been discussed.

Calcium-enhanced aggregation of serum amyloid P component and its inhibition by the ligands heparin and heparan sulphate

Serum amyloid P component (SAP) is a pentraxin found in the circulation and in all forms of amyloid deposits. Its physiological and pathophysiological functions are largely unknown. Electron microscopy showed purified human SAP to consist of double pentameric discs compatible with the results of size chromatography. The formation of double pentamers did not require calcium ions. The outer diameter of the discs arranged face‐to‐face was 11.6 nm and the inner diameter 3.2 nm. The thickness of single and double pentamers was 4.1 and 8.7 nm, respectively. Quadruple pentamers were occasionally seen. The self‐aggregation of human SAP molecules was investigated in the presence and absence of calcium ions at different concentrations. In calcium‐free solutions few and mostly small SAP aggregates were seen. After addition of calcium at increasing concentration the aggregates grew in size and crystallinelike structures were formed already at 2 mM calcium. At 25 mM calcium, large aggregates with a crystalline array occasionally exhibiting cylinders predominated. Binding of the ligands heparin and heparan sulphate to SAP completely abolished the calcium‐enhanced aggregation, but the distribution of the SAP molecules was affected, resulting in strands or groups of adjacent molecules. The electrophoretic mobility of SAP was moreover significantly altered after its calcium‐dependent reaction with these ligands. We conclude that purified SAP has a tendency to double pentamer formation and self‐aggregation also in the absence of calcium ions. However, aggregation is greatly enhanced even at low concentrations (2 mM) of calcium. SAPs tendency to self‐aggregation is abolished after its binding to heparin or heparan sulphate. Furthermore, our TEM studies indicate that purified human SAP freed of its natural ligands has the double pentameric form, whereas the electrophoretic investigations suggest that SAPs interaction with low‐molecular‐weight natural ligands in serum prevents homodimerization and self‐aggregation.

Multiple isoforms of the human pentraxin serum amyloid P component

Human serum amyloid P component (SAP) isolated from 20 healthy individuals was analyzed by anion exchange chromatography and isoelectric focusing (IEF) in order to investigate the existence of multiple forms of SAP and interindividual structural differences. Anion exchange chromatography showed one major and several minor subpopulations of SAP. IEF of all SAP isolates showed a previously unreported degree of heterogeneity with six isoelectric forms (pKi range 5.5-6.1) and with minor interindividual differences in respect of isoelectric points. Total enzymatic deglycosylation of SAP reduced the number of bands in IEF to two indicating the existence of two types of polypeptide chains.

Ultrastructure of psoriatic epidermis

The ultrastructure of human affected and unaffected psoriatic epidermis was studied in skin biopsies from 5 patients and 3 normal controls. Transmission electron microscopic investigations revealed abnormalities in all cell layers of the affected epidermis. Common to psoriatic keratinocytes from affected epidermis was the reduction of tonofilaments. The essential ultrastructural changes were located in the stratum granulosum and stratum corneum. Thus, abscence of the fusion between the keratohyalin granules and the tonofilaments was found in stratum granulosum. The keratinocytes of the stratum corneum showed a large accumulation of ribosomes and vesicles resembling lipid vesicles.