Amino acid sequence of human factor D of the complement system: Similarity in sequence between factor D and proteases of non-plasma origin

The amino acid sequence of human factor D is proposed from the analysis of the peptides produced by treatment of the factor D with cyanogen bromide, iodosobenzoic acid, trypsin and V-8 protease. Comparison of the proposed sequence with the sequences of other serine esterases indicated that factor D, although it is a plasma serine esterase, is more closely related to certain proteases not found in the plasma than to other plasma serine esterases of the complement system. For example, 36% and 32% identity in amino acid sequence was found on comparison of factor D with elastase and group-specific protease, respectively. Whereas only 27% and 18% identity was observed between factor D and the other complement serine esterases, Clr and factor B, respectively.     

Inhibition of the alternative pathway of human complement by structural analogues of sialic acid

Liposomes were used to determine whether gangliosides containing certain structurally defined analogues of sialic acid could inhibit activation of the alternative pathway of human C. Gangliosides containing sialic acid residues with modifications in the N-acetyl group, carboxyl group, or polyhydroxylated tail were either isolated from natural sources or prepared by chemical modification of the native sialic acid structure. Sialic acid lost more than 90% of its inhibitory activity after removal of just the C9 carbon from the polyhydroxylated tail. Sialic acid was also unable to inhibit activation after converting the carboxyl group to a hydroxymethyl group. Galactose oxidase/NaB3H4 treatment of liposomes containing gangliosides with native or modified sialic acid residues confirmed that neither modification altered the amount of gangliosides exposed at the liposome surface. Changing the N-linked acetyl group to a glycolyl group had no effect on the inhibitory activity of sialic acid. These data further define the structural features of sialic acid that are important in regulation of alternative pathway activation. Both the C9 carbon of the polyhydroxylated tail and the carboxyl group are essential for this function; whereas, the N-linked acetyl group may be modified without loss of activity.     

Inhibiting alternative pathway complement activation by targeting the factor D exosite

By virtue of its amplifying property, the alternative complement pathway has been implicated in a number of inflammatory diseases and constitutes an attractive therapeutic target. An anti-factor D Fab fragment (AFD) was generated to inhibit the alternative complement pathway in advanced dry age-related macular degeneration. AFD potently prevented factor D (FD)-mediated proteolytic activation of its macromolecular substrate C3bB, but not proteolysis of a small synthetic substrate, indicating that AFD did not block access of the substrate to the catalytic site. The crystal structures of AFD in complex with human and cynomolgus FD (at 2.4 and 2.3 Å, respectively) revealed the molecular details of the inhibitory mechanism. The structures show that the AFD-binding site includes surface loops of FD that form part of the FD exosite. Thus, AFD inhibits FD proteolytic function by interfering with macromolecular substrate access rather than by inhibiting FD catalysis, providing the molecular basis of AFD-mediated inhibition of a rate-limiting step in the alternative complement pathway.     

Amino acid sequence of the human fibronectin receptor

The amino acid sequence deduced from cDNA of the human placental fibronectin receptor is reported. The receptor is composed of two subunits: an alpha subunit of 1,008 amino acids which is processed into two polypeptides disulfide bonded to one another, and a beta subunit of 778 amino acids. Each subunit has near its COOH terminus a hydrophobic segment. This and other sequence features suggest a structure for the receptor in which the hydrophobic segments serve as transmembrane domains anchoring each subunit to the membrane and dividing each into a large ectodomain and a short cytoplasmic domain. The alpha subunit ectodomain has five sequence elements homologous to consensus Ca2+-binding sites of several calcium-binding proteins, and the beta subunit contains a fourfold repeat strikingly rich in cysteine. The alpha subunit sequence is 46% homologous to the alpha subunit of the vitronectin receptor. The beta subunit is 44% homologous to the human platelet adhesion receptor subunit IIIa and 47% homologous to a leukocyte adhesion receptor beta subunit. The high degree of homology (85%) of the beta subunit with one of the polypeptides of a chicken adhesion receptor complex referred to as integrin complex strongly suggests that the latter polypeptide is the chicken homologue of the fibronectin receptor beta subunit. These receptor subunit homologies define a superfamily of adhesion receptors. The availability of the entire protein sequence for the fibronectin receptor will facilitate studies on the functions of these receptors.     

The central role of the alternative complement pathway in human disease

The complement system is increasingly recognized as important in the pathogenesis of tissue injury in vivo following immune, ischemic, or infectious insults. Within the complement system, three pathways are capable of initiating the processes that result in C3 activation: classical, alternative, and lectin. Although the roles that proinflammatory peptides and complexes generated during complement activation play in mediating disease processes have been studied extensively, the relative contributions of the three activating pathways is less well understood. Herein we examine recent evidence that the alternative complement pathway plays a key and, in most instances, obligate role in generating proinflammatory complement activation products in vivo. In addition, we discuss new concepts regarding the mechanisms by which the alternative pathway is activated in vivo, as recent clinical findings and experimental results have provided evidence that continuous active control of this pathway is necessary to prevent unintended targeting and injury to self tissues.     

Amino acid sequence of the trypsin-generated C3d fragment from human complement factor C3.

Energy metabolism in proliferating cultured rat thymocytes was compared with that of freshly prepared non-proliferating resting cells. Cultured rat thymocytes enter a proliferative cycle after stimulation by concanavalin A and Lymphocult T (interleukin-2), with maximal rates of DNA synthesis at 60 h. Compared with incubated resting thymocytes, glucose metabolism by incubated proliferating thymocytes was 53-fold increased; 90% of the amount of glucose utilized was converted into lactate, whereas resting cells metabolized only 56% to lactate. However, the latter oxidized 27% of glucose to CO2, as opposed to 1.1% by the proliferating cells. Activities of hexokinase, 6-phosphofructokinase, pyruvate kinase and aldolase in proliferating thymocytes were increased 12-, 17-, 30- and 24-fold respectively, whereas the rate of pyruvate oxidation was enhanced only 3-fold. The relatively low capacity of pyruvate degradation in proliferating thymocytes might be the reason for almost complete conversion of glucose into lactate by these cells. Glutamine utilization by rat thymocytes was 8-fold increased during proliferation. The major end products of glutamine metabolism are glutamate, aspartate, CO2 and ammonia. A complete recovery of glutamine carbon and nitrogen in the products was obtained. The amount of glutamate formed by phosphate-dependent glutaminase which entered the citric acid cycle was enhanced 5-fold in the proliferating cells: 76% was converted into 2-oxoglutarate by aspartate aminotransferase, present in high activity, and the remaining 24% by glutamate dehydrogenase. With resting cells the same percentages were obtained (75 and 25). Maximal activities of glutaminase, glutamate dehydrogenase and aspartate aminotransferase were increased 3-, 12- and 6-fold respectively in proliferating cells; 32% of the glutamate metabolized in the citric acid cycle was recovered in CO2 and 61% in aspartate. In resting cells this proportion was 41% and 59% and in mitogen-stimulated cells 39% and 65% respectively. Addition of glucose (4 mM) or malate (2 mM) strongly decreased the rates of glutamine utilization and glutamate conversion into 2-oxoglutarate by proliferating thymocytes and also affected the pathways of further glutamate metabolism. Addition of 2 mM-pyruvate did not alter the rate of glutamine utilization by proliferating thymocytes, but decreased the rate of metabolism beyond the stage of glutamate significantly. Formation of acetyl-CoA in the presence of pyruvate might explain the relatively enhanced oxidation of glutamate to CO2 (56%) by proliferating thymocytes.     

Amino acid sequence around the thiol and reactive acyl groups of human complement component C4.

Activation of the fourth component of complement (C4) by C1s results in the generation of a reactive acyl group, able to react with putrescine, and in the release of a free thiol group that cannot be detected in the native haemolytically active molecule. Both the reactive acyl group and the free thiol group have been shown to reside in C4d, a fragment of the alpha'-chain of C4b derived from digestion of the molecule with the control proteins C3b inactivator and C4-binding protein. Peptides derived from CNBr digestion of [1,4-14C]putrescine-labelled and iodo(2-14C]acetic acid-labelled C4d have been obtained and used to establish a continuous sequence of 88 residues from the N-terminus of the molecule. The thiol and reactive acyl groups are contained in an octapeptide that shows near identity with the equivalent sequences reported for alpha 2-macroglobulin and C3. Other adjacent short sections also show homology of sequence between the three proteins, and it is highly likely that they contribute to the overall structure that gives a unique reactivity to the thiol ester bond postulated to exist in the native forms of the three proteins.     

The alternative complement pathway revisited

Alternative pathway amplification plays a major role for the final effect of initial specific activation of the classical and lectin complement pathways, but the quantitative role of the amplification is insufficiently investigated. In experimental models of human diseases in which a direct activation of alternative pathway has been assumed, this interpretation needs revision placing a greater role on alternative amplification. We recently documented that the alternative amplification contributed to 80-90% of C5 activation when the initial activation was highly specific for the classical pathway. The recent identification of properdin as a recognition factor directly initiating alternative pathway activation, like C1q in the classical and mannose-binding lectin in the lectin pathway initiates a renewed interest in the reaction mechanisms of complement. Complement and Toll-like receptors, including the CD14 molecule, are two main upstream recognition systems of innate immunity, contributing to the inflammatory reaction in a number of conditions including ischemia-reperfusion injury and sepsis. These systems act as "double-edged swords", being protective against microbial invasion, but harmful to the host when activated improperly or uncontrolled. Combined inhibition of complement and Toll-like receptors/CD14 should be explored as a treatment regimen to reduce the overwhelming damaging inflammatory response during sepsis. The alternative pathway should be particularly considered in this regard, due to its uncontrolled amplification in sepsis. The alternative pathway should be regarded as a dual system, namely a recognition pathway principally similar to the classical and lectin pathways, and an amplification mechanism, well known, but quantitatively probably more important than generally recognized.     

Activation of complement via the alternative pathway

Activation of complement via the alternative pathway represents one means of natural resistance to infection because it is capable of neutralizing a wide variety of potential pathogens in the total absence of antibody. The pathway involves six serum proteins and possesses a unique amplification system capable of depositing large numbers of C3b molecules on the surfaces of activating particles. C3b deposition enhances phagocytosis and results in activation of the membrane attack pathway of complement. C3b attachment is covalent, arising from a reaction between an intramolecular thiolester bond in nascent C3b and nucleophiles such as hydroxyl groups on surface carbohydrates. The reactions that initiate C3b attachment are not specific interactions like those initiating other biological cascade systems, but involve slow, spontaneous hydrolysis of the thiolester bond in C3 and subsequent random deposition of C3b onto all nearby surfaces. Once bound, C3b is capable of discriminating between host-derived cells and activating particles. Recognition is evidenced by a lower affinity between activator-bound C3b and the complement control protein factor H. Measurements of the association constant between unbound, soluble C3b and factor H suggest that activator-bound C3b recognizes structures on activators that inhibit factor H binding.     

Effect of gangliosides on activation of the alternative pathway of human complement

Liposomes as defined model membranes were used to quantitatively study the effects of specific sialic acid containing glycolipids on activation of the alternative pathway of human C. Liposomes containing dimyristoylphosphatidylethanolamine, cholesterol, and cerebrosides at molar ratios of 1.0/0.75/0.33 activated the alternative pathway in human serum treated with MgEGTA. Activation was measured by C3 conversion and the deposition of total C3 and functional C3b on the liposome surface. The monosialoganglioside GM1, when incorporated into the activating liposome membrane at molar ratios between 10(-5) and 10(-2), inhibited activation in a dose-dependent manner. Sialosylparagloboside also inhibited activation in human serum, and inhibition was completely reversed after neuraminidase treatment. The degree of inhibition by GM1 correlated with the relative amount of GM1 exposed on the liposome surface. Sialic acid did not directly inhibit the binding of C3b when liposomes containing gangliosides were incubated with the purified components C3, B, D, and P. GM1 did inhibit activation when liposomes were incubated with a mixture of purified C3, B, D, P, H, and I. Binding assays with radiolabeled H showed increased binding of H to liposome-bound C3b in the presence of GM1. These results establish the ability of sialic acid on glycolipids to promote H binding to C3b and thereby regulate alternative pathway activation on a defined lipid membrane.     

Activation of the alternative complement pathway by isolated human glomerular basement membrane

The capacity of isolated human glomerular basement membrane (GBM) to initiate surface activation of the human alternative complement pathway was defined by the deposition of C3b under circumstances in which the classical complement pathway was inoperative. The deposition of C3b from normal or C2-deficient serum was time- and magnesium-dependent, implying a role for the alternative pathway. Normal human serum rendered deficient in D did not sustain C3b deposition until its reconstitution with D, indicating an absolute requirement for a protein unique to the alternative pathway and essential to the cleavage activation of the C3 amplification convertase of that pathway. The capacity of the excess control proteins H and I to prevent C3b deposition onto GBM incubated in C2-deficient serum provided further evidence for the direct activation of the alternative pathway in this system. The use of radiolabeled monoclonal antibody to localize the deposited C3b afforded specificity and quantitation of about 100 ng of C3b/mg of GBM. Immunohistochemical analysis with a monoclonal antibody to detect C3b demonstrated its deposition to be confined to the epithelial surface of the GBM.     

Activation of the alternative pathway of complement by human peripheral nerve myelin

Destruction of peripheral nerve myelin (PNM) occurs as a consequence of a variety of pathologic conditions affecting the peripheral nervous system. In certain primary demyelinating neuropathies, several lines of evidence implicate complement in the pathogenesis of demyelination. In this study we demonstrate that human PNM consumes complement in vitro in the absence of specific antibody or C1 activation. Furthermore, activation of complement by PNM via the alternative pathway was shown by cleavage of C3 in normal human serum (NHS) and of B in C2-deficient serum (C2d-HS). Increasing consumption of hemolytic activity of C3 in Mg-EGTA-treated NHS was also noted with increasing amounts of PNM. Pronase treatment of PNM abolished C3 consumption, suggesting that a protein component exposed on the surface of myelin participated in the alternative pathway activation. When P0, the major amphiphilic glycoprotein of PNM, was incorporated into artificial lipid bilayers, the Po-liposomes consumed C3 activity in NHS containing Mg-EGTA. Pronase treatment of Po-liposomes abolished C3 consumption to the level of control liposomes, indicating that P0 was responsible for at least part of the activation seen with peripheral myelin.     

Amino acid sequence of galactosamine-containing glycopeptides in the hinge region of a human immunoglobulin D

Amino acid sequence and the location of seven galactosamine oligosaccharide moieties of the hinge region of the δ chain of human IgD NIG-65 have been determined. These oligosaccharide moieties are distributed in two distinct fashions: 1) three clusters each consisting of five amino acid residues with two consecutive attachment sites either Ala-X-Ala-Ser-Ser or Ala-X-Ala-Thr-Thr, where X can be any amino acid including proline, 2) one triplet sequence Val-Pro-Thr with one attachment site. We propose two rules with regard to the acceptor sequence for galactosamine oligosaccharides, the quintet sequence rule and triplet sequence rule.     

Amino acid sequence of human liver cathepsin B

The complete amino acid sequence of cathepsin B (EC 3.4.22.1) from human liver was determined. The 252-residue sequence was obtained by automated solid-phase Edman degradation of the light and heavy chain resulting from limited proteolysis of the single-chain enzyme and of fragments produced by cyanogen bromide and enzymatic cleavage of the heavy chain. Human liver cathepsin B has 83.7% identical residues with the corresponding enzyme from rat liver. Comparison of both mammalian cathepsin B sequences with the sequence of papain provides further evidence that lysosomal and plant cysteine proteinases have evolved from a common ancestor and share a similar catalytic mechanism.     

Amino acid sequence of human von Willebrand factor

The complete amino acid sequence of human von Willebrand factor (vWF) is presented. Most of the sequence was determined by analysis of the S-carboxymethylated protein. Some overlaps not provided by the protein sequence analysis were obtained from the sequence predicted by the nucleotide sequence of a cDNA clone [Sadler, J.E., Shelton-Inloes, B.B., Sorace, J., Harlan, M., Titani, K., & Davie, E.W. (1985) Proc. Natl. Acad. Sci. U.S.A. 82, 6391-6398]. The protein is composed of 2050 amino acid residues containing 12 Asn-linked and 10 Thr/Ser-linked oligosaccharide chains. One of the carbohydrate chains is linked to an Asn residue in the sequence Asn-Ser-Cys rather than the usual Asn-X-Ser/Thr sequence. The sequence of von Willebrand factor includes several regions bearing evidence of internal gene duplication of ancestral sequences. The protein also contains the tetrapeptide sequence Arg-Gly-Asp-Ser (at residues 1744-1747), which may be a cell attachment site, as in fibronectin. The amino- and carboxyl-terminal regions of the molecule contain clusters of half-cystinyl residues. The sequence is unique except for some homology to human complement factor B.