Phosphorylcholine acts as a Ca2+-dependent receptor molecule for lymphocyte perforin

Large granular lymphocytes and cytolytic T-lymphocytes (CTL) contain numerous cytoplasmic granules thought to be responsible, at least in part, for the cytolytic activity of these effector cells. Isolated granules are lytic for a variety of target cells and the granule proteins are specifically released upon target-cell interaction. Major proteins in mouse CTL granules are a family of seven serine proteases designated granzymes A to G, and a pore-forming protein called perforin (cytolysin). Purified perforin is cytolytic in the presence of Ca2+ and shows ultrastructural, immunological and amino-acid sequence similarities to complement component C9. Despite these similarities, perforin and C9 are clearly distinct in their mode of target-cell recognition. Whereas C9 insertion is absolutely dependent on a receptor moiety assembled from the complement proteins C5b, C6, C7, and C8 on the target-cell membrane, no requirement for a receptor molecule has been reported for perforin. Here, we demonstrate that phosphorylcholine acts as a specific, Ca2+-dependent receptor molecule for perforin.     

Structural/functional similarity between proteins involved in complement- and cytotoxic T-lymphocyte-mediated cytolysis

Cytolysis mediated by complement or cytolytic lymphocytes results in the formation of morphology similar lesions in the target membrane. These lesions, formed by the polymerization of C9 or perforin respectively, contribute the major killing action by causing osmotic lysis of the target cell. Following the suggestion of Mayer that the mechanisms of humoral and cell-mediated cytotoxicity might be related, studies into the morphology of the membrane lesions formed, and the proteins responsible for causing the lesions, have shown several similarities. While the lesion caused by natural and T-killer cells is a little larger than that caused by complement, its overall shape is similar and in both cases the cylindrical pore is formed by polymerization of a monomeric subunit, C9 (relative molecular mass, Mr = 71,000) for complement, and perforin (Mr = 66,000) for cell-mediated cytotoxicity. C9 has an absolute requirement for a receptor in the target membrane formed by the earlier membrane attack complex components, C5b, C6, C7 and C8 (ref. 8). For perforin, polymerization in a target membrane requires no receptor, specificity being derived from the specific recognition between killer and target cell. Both proteins can be made to polymerize in vitro by the addition of divalent cations (Zn2+ for C9 (ref. 16) and Ca2+ for perforin) and the resultant complexes closely resemble their physiological counterparts. Antibodies raised against lymphocyte-killed targets have also been shown to cross-react with complement proteins, but the antigenically related proteins were not determined in these studies. We show here using purified proteins that perforin, C9 and complexes involving C7 and C8 share a common antigenic determinant which is probably involved in polymerization.     

人穿孔素羧基端肽段的表达纯化与活性鉴定

穿孔素,即成孔蛋白（pore forming protein,PFP),其溶细胞作用与免疫调节和自身免疫病以及其它多种疾病过程中的免疫性病理损伤相关。为得到足够量的PFP建立与之相关的免疫学研究手段用于基础和临床研究,在已克隆人PFP cDNA的基础上,用基因工程方法表达了人PFP C端124个氨基酸肽段（hPFP-C),并通过谷胱甘肽琼脂糖新和层析获得纯化的GST/hPFP-C融合蛋白,经凝血酶酶切和再次北极和层析去除GST部分,得到了纯化的hPFP-C蛋白。纯化的hPFP-C蛋白与兔红细胞共育,呈现钙依赖的溶血活性。     

Cytotoxic T lymphocytes recognize influenza haemagglutinin that lacks a signal sequence

A surprising feature of most cytotoxic T lymphocytes (CTL) responding to influenza infection is that they recognize the unglycosylated (non-transmembrane) proteins of the virus, including the nucleoprotein. Recognition of cells that express nucleoprotein by CTL does not depend on a definite signal sequence within the protein, and the epitopes recognized can be defined with short synthetic peptides in vitro. Haemagglutinin (HA), the major transmembrane protein of the virus, is recognized by a minor population of CTL from infected mice. We have deleted the sequence coding for the N-terminal signal peptide from a complementary DNA encoding HA of the H1 subtype. The signal-deleted HA is detected with antibodies as a short-lived, unglycosylated, intracellular protein. However, CTL raised to the complete molecule recognize cells expressing the signal-deleted HA and vice versa. These results cast doubts on the assumption that CTL recognize the HA molecule only after its insertion into the plasma membrane.     

An H+-ATPase in opposite plasma membrane domains in kidney epithelial cell subpopulations

Vectorial solute transport by epithelia requires the polarized insertion of transport proteins into apical or basolateral plasmalemmal domains. In the specialized intercalated cells of the kidney collecting duct, the selective placement of an apical plasma membrane proton-pumping ATPase (H+-ATPase) and of a basolateral membrane anion-exchange protein results in transepithelial proton secretion. It is currently believed that amino-acid sequences of membrane proteins contain critical signalling regions involved in sorting these proteins to specific membrane domains. Recently, it was proposed that intercalated cells can reverse their direction of proton secretion under different acid-base conditions by redirecting proton pumps from apical to basolateral membranes, and anion exchangers from basolateral to apical membranes. But others have found that antibodies raised against the red cell anion-exchange protein (Band 3) only labelled intercalated cells at the basolateral plasma membrane, providing evidence against the model of polarity reversal. In this report, we have examined directly the distribution of proton pumps in kidney intercalated cells using specific polyclonal antibodies against subunits of a bovine kidney medullary H+-ATPase. We find that some cortical collecting duct intercalated cells have apical plasma membrane proton pumps, whereas others have basolateral pumps. This is the first direct demonstration of neighbouring epithelial cells maintaining opposite polarities of a transport protein. Thus, either subtle structural differences exist between proton pumps located at opposite poles of the cell, or factors other than protein sequence determine the polarity of H+-ATPase insertion.     

Trichosanthin can spontaneously penetrate phospholipid monolayer under acid condition

Tianhuafen is a kind of traditional Chinese medicine for abortion, which has long been used in China. The basic protein trichosanthin (TCS) is responsible for such activity. As a ribosome inactivating protein (RIP), trichosanthin removes A4304 in the 28s rRNA via the N-glycosidase activity and inactivates the ribosome. However, it remains unclear how TCS can enter the cytoplasm. In the experiment, monolayers at air/water interface have been used to study the interaction between TCS and phospholipid membrane. The results show that TCS can penetrate negatively charged phospholipid monolayer under acid condition, and its membrane penetrating ability is obviously pH-dependent. A hypothesis of TCS penetrating biological membrane under acid condition is proposed based on the obtained result.     

Interaction of SynaptotagminⅠ with Phospholipid Membrane: A Monolayer Study

IntroductionSynaptotagmin is a family of vesicletransmembrane proteins present in synapticvesicles and large secretary granules of neuronsand endocrine cells[1 ] .It is a major constituent ofsynaptic vesicle membranes,comprising7% 8%of the total vesicle protein,characterized by ashort intravesiclar N-terminus,a singletransmembrane region,and a long plasmicdomain.　The best-charaterized form of synaptotagmin,syt ,is found abundantly in rostrol brain.Syt was first described in 1 981 [2 ] ,and i…     

A 'molten-globule' membrane-insertion intermediate of the pore-forming domain of colicin A

The 'molten' globular conformation of a protein is compact with a native secondary structure but a poorly defined tertiary structure. Molten globular states are intermediates in protein folding and unfolding and they may be involved in the translocation or insertion of proteins into membranes. Here we investigate the membrane insertion of the pore-forming domain of colicin A, a bacteriocin that depolarizes the cytoplasmic membrane of sensitive cells. We find that this pore-forming domain, the insertion of which depends on pH, undergoes a native to molten globule transition at acidic pH. The variation of the kinetic constant of membrane insertion of the protein into negatively charged lipid vesicles as a function of the interfacial pH correlates with the appearance of the acidic molten globular state, indicating that this state could be an intermediate formed during the insertion of colicin A into membranes.     

Homology of perforin to the ninth component of complement (C9)

Perforin is one of the cytolytic factors present in the cytoplasmic granules of mouse cytotoxic T lymphocytes and natural killer cells. We have determined the sequence of the N-terminal amino acids of perforin purified from a mouse natural killer cell line, and, by using oligonucleotide probes corresponding to the amino acid residues, we have identified a complementary DNA encoding perforin from the cDNA library of a mouse cytotoxic T lymphocyte clone. As predicted from the functional similarities between perforin and the ninth component of the serum cytolytic system, complement (C9) (refs 4-8), the deduced primary structure of perforin has homology with C9 at their respective functionally conserved regions. We find that perforin is only expressed in killer cell lines, and not in helper T lymphocytes or other tumour cells tested. Thus we have provided direct molecular evidence that a killer-cell-specific protein evolutionally linked to C9 is involved in cell-mediated cytolysis.     

A substrate-specific inhibitor of protein translocation into the endoplasmic reticulum

The segregation of secretory and membrane proteins to the mammalian endoplasmic reticulum is mediated by remarkably diverse signal sequences that have little or no homology with each other. Despite such sequence diversity, these signals are all recognized and interpreted by a highly conserved protein-conducting channel composed of the Sec61 complex. Signal recognition by Sec61 is essential for productive insertion of the nascent polypeptide into the translocation site, channel gating and initiation of transport. Although subtle differences in these steps can be detected between different substrates, it is not known whether they can be exploited to modulate protein translocation selectively. Here we describe cotransin, a small molecule that inhibits protein translocation into the endoplasmic reticulum. Cotransin acts in a signal-sequence-discriminatory manner to prevent the stable insertion of select nascent chains into the Sec61 translocation channel. Thus, the range of substrates accommodated by the channel can be specifically and reversibly modulated by a cell-permeable small molecule that alters the interaction between signal sequences and the Sec61 complex.     

The mechanism of Z alpha 1-antitrypsin accumulation in the liver.

Most northern Europeans have only the normal M form of the plasma protease inhibitor alpha 1-antitrypsin, but some 4% are heterozygotes for the Z deficiency variant. For reasons that have not been well-understood, the Z mutation results in a blockage in the final stage of processing of antitrypsin in the liver such that in the Z homozygote only 15% of the protein is secreted into the plasma. The 85% of the alpha 1-antitrypsin that is not secreted accumulates in the endoplasmic reticulum of the hepatocyte; much of it is degraded but the remainder aggregates to form insoluble intracellular inclusions. These inclusions are associated with hepatocellular damage, and 10% of newborn Z homozygotes develop liver disease which often leads to a fatal childhood cirrhosis. Here we demonstrate the molecular pathology underlying this accumulation and describe how the Z mutation in antitrypsin results in a unique molecular interaction between the reactive centre loop of one molecule and the gap in the A-sheet of another. This loop-sheet polymerization of Z antitrypsin occurs spontaneously at 37 degrees C and is completely blocked by the insertion of a specific peptide into the A-sheet of the antitrypsin molecule. Z antitrypsin polymerized in vitro has identical properties and ultrastructure to the inclusions isolated from hepatocytes of a Z homozygote. The concentration and temperature dependence of this loop-sheet polymerization has implications for the management of the liver disease of the newborn Z homozygote.     

Monoclonal antibodies demonstrate protection of polymorphonuclear leukocytes against complement attack

Studies on erythrocytes have shown that the formation of the membrane attack complex on a cell surface inevitably results in lysis. However, it is known that nucleated cells are much more difficult to kill with complement, although the molecular basis of this resistance has never been established. We have shown that a very early intracellular event, occurring within seconds of formation of the attack complex in the membrane, is a rise in cytoplasmic Ca2+, which can activate cell responses without cell death 5,6. Here we report the use of a monoclonal antibody to the terminal complement component C9, quantified by 125I and visualized by fluorescein, to demonstrate a protection mechanism in polymorphonuclear leukocytes (PMNs) attacked by complement, involving removal of the attack complex by vesiculation. Concomitantly, there is a Ca2+-dependent activation of reactive oxygen metabolite production without cell lysis. These findings have important implications in the evolutionary and pathological significance of the terminal components of the complement pathway.     

The crystal structure of diphtheria toxin.

The crystal structure of the diphtheria toxin dimer at 2.5 A resolution reveals a Y-shaped molecule of three domains. The catalytic domain, called fragment A, is of the alpha + beta type. Fragment B actually consists of two domains. The transmembrane domain consists of nine alpha-helices, two pairs of which are unusually apolar and may participate in pH-triggered membrane insertion and translocation. The receptor-binding domain is a flattened beta-barrel with a jelly-roll-like topology. Three distinct functions of the toxin, each carried out by a separate structural domain, can be useful in designing chimaeric proteins, such as immunotoxins, in which the receptor-binding domain is substituted with antibodies to target other cell types.     

The structural basis for membrane binding and pore formation by lymphocyte perforin

Natural killer cells and cytotoxic T lymphocytes accomplish the critically important function of killing virus-infected and neoplastic cells. They do this by releasing the pore-forming protein perforin and granzyme proteases from cytoplasmic granules into the cleft formed between the abutting killer and target cell membranes. Perforin, a 67-kilodalton multidomain protein, oligomerizes to form pores that deliver the pro-apoptopic granzymes into the cytosol of the target cell. The importance of perforin is highlighted by the fatal consequences of congenital perforin deficiency, with more than 50 different perforin mutations linked to familial haemophagocytic lymphohistiocytosis (type 2 FHL). Here we elucidate the mechanism of perforin pore formation by determining the X-ray crystal structure of monomeric murine perforin, together with a cryo-electron microscopy reconstruction of the entire perforin pore. Perforin is a thin 'key-shaped' molecule, comprising an amino-terminal membrane attack complex perforin-like (MACPF)/cholesterol dependent cytolysin (CDC) domain followed by an epidermal growth factor (EGF) domain that, together with the extreme carboxy-terminal sequence, forms a central shelf-like structure. A C-terminal C2 domain mediates initial, Ca(2+)-dependent membrane binding. Most unexpectedly, however, electron microscopy reveals that the orientation of the perforin MACPF domain in the pore is inside-out relative to the subunit arrangement in CDCs. These data reveal remarkable flexibility in the mechanism of action of the conserved MACPF/CDC fold and provide new insights into how related immune defence molecules such as complement proteins assemble into pores.     

Structure and function of human perforin

Perforin (P1) is a cytolytic protein with similarity to complement component C9. P1 has been described as a unique component of murine cytolytic T-cell and rat natural killer cell granules Previous studies indicated that human granules and P1 differed from murine granules and P1 in that they appeared to be cytolytically less active and lacked the haemolytic activity characteristic of P1. It has been suggested that P1, like C9, is under the control of the homologous restriction factor. Here we determine the primary structure of human P1, re-examine its functional properties, and address the question of homologous restriction.     

Structure of the membrane-pore-forming fragment of colicin A

Colicins are antibiotic proteins produced by and active against sensitive Escherichia coli and closely related bacteria. They can adsorb to specific receptors located at the external surface of the outer membrane of sensitive cells, and are then translocated to their specific targets within these cells. The largest group of colicins comprises those which can form voltage-dependent channels in membranes, thereby destroying the cell's energy potential. Colicin molecules are organized in structural domains, each domain carrying one function associated with the toxin's lethal activity. The pore-forming activity seems to be located at the carboxyl terminus. A thermolytic fragment comprising amino acids 389-592 from colicin A has pore-forming properties very similar to those of the entire molecule. This fragment is soluble in aqueous medium and spontaneously inserts into lipid bilayers. We have determined the structure of the pore-forming fragment of colicin A by X-ray crystallography and refinement at 2.5 A resolution. The protein consists of ten alpha-helices organized in a three-layer structure. Two of the helices are completely buried within the structure and form a hydrophobic hairpin loop similar to that proposed for signal sequences which function in translocation. We present a model for insertion of the protein into lipid bilayers the features of which may be applicable in other biological systems involving protein insertion or translocation across membranes.     

新型嵌合补体抑制分子HCI-3的设计构建及真核细胞表达的研究

目的：基于抑制补体活化的两个关键环节，即C3／C5转化酶及MAC的形成，设计、构建及制备一种新型、高效的补体抑制剂．方法：首先设计引物，通过PCR技术扩增重组可溶性CD46／CD55／CD59嵌合分子cDNA片段，重组于pcDNA3．1( )真核表达载体上，构建兼有三分子功能的CD46／CD55／CD59嵌合补体抑制分子，命名为HCI-3(Human Chimeric Complement Inhibitor,HCI-3)．分别利用COS-7细胞和CHO细胞进行表达，并用抗人CDl6多抗及抗人CD55，CD59单抗对表达产物进行Western Blotting鉴定．结果：DNA测序结果证实，前端带有CD59信号肽序列，后端融合有编码6个组氨酸碱基的HCl-3 cDNA片段的阅读框完整．免疫印迹结果显示，表达的重组蛋白分别能与抗人CD46多克隆抗体和抗人CD55，CD59单抗结合．结论：成功构建并在真核细胞内表达了重组可溶性HCl-3分子，为进一步研制和开发新一代多功能、多靶点补体抑制剂奠定了基础．     

IRSp53 is an essential intermediate between Rac and WAVE in the regulation of membrane ruffling

Neural Wiskott-Aldrich syndrome protein (N-WASP) functions in several intracellular events including filopodium formation, vesicle transport and movement of Shigella frexneri and vaccinia virus, by stimulating rapid actin polymerization through the Arp2/3 complex. N-WASP is regulated by the direct binding of Cdc42 (refs 7, 8), which exposes the domain in N-WASP that activates the Arp2/3 complex. A WASP-related protein, WAVE/Scar, functions in Rac-induced membrane ruffling; however, Rac does not bind directly to WAVE, raising the question of how WAVE is regulated by Rac. Here we demonstrate that IRSp53, a substrate for insulin receptor with unknown function, is the 'missing link' between Rac and WAVE. Activated Rac binds to the amino terminus of IRSp53, and carboxy-terminal Src-homology-3 domain of IRSp53 binds to WAVE to form a trimolecular complex. From studies of ectopic expression, we found that IRSp53 is essential for Rac to induce membrane ruffling, probably because it recruits WAVE, which stimulates actin polymerization mediated by the Arp2/3 complex.     

Targeted recycling of PECAM from endothelial surface-connected compartments during diapedesis

Leukocytes enter sites of inflammation by squeezing through the borders between endothelial cells that line postcapillary venules at that site. This rapid process, called transendothelial migration (TEM) or diapedesis, is completed within 90 s after a leukocyte arrests on the endothelial surface. In this time, the leukocyte moves in ameboid fashion across the endothelial borders, which remain tightly apposed to it during transit. It is not known how the endothelial cell changes its borders rapidly and reversibly to accommodate the migrating leukocyte. Here we show that there is a membrane network just below the plasmalemma at the cell borders that is connected at intervals to the junctional surface. PECAM-1, an integral membrane protein with an essential role in TEM, is found in this compartment and constitutively recycles evenly along endothelial cell borders. During TEM, however, recycling PECAM is targeted to segments of the junction across which monocytes are in the act of migration. In addition, blockade of TEM with antibodies against PECAM specifically blocks the recruitment of this membrane to the zones of leukocyte migration, without affecting the constitutive membrane trafficking.     

The structure of complement C3b provides insights into complement activation and regulation

The human complement system is an important component of innate immunity. Complement-derived products mediate functions contributing to pathogen killing and elimination. However, inappropriate activation of the system contributes to the pathogenesis of immunological and inflammatory diseases. Complement component 3 (C3) occupies a central position because of the manifold biological activities of its activation fragments, including the major fragment, C3b, which anchors the assembly of convertases effecting C3 and C5 activation. C3 is converted to C3b by proteolysis of its anaphylatoxin domain, by either of two C3 convertases. This activates a stable thioester bond, leading to the covalent attachment of C3b to cell-surface or protein-surface hydroxyl groups through transesterification. The cleavage and activation of C3 exposes binding sites for factors B, H and I, properdin, decay accelerating factor (DAF, CD55), membrane cofactor protein (MCP, CD46), complement receptor 1 (CR1, CD35) and viral molecules such as vaccinia virus complement-control protein. C3b associates with these molecules in different configurations and forms complexes mediating the activation, amplification and regulation of the complement response. Structures of C3 and C3c, a fragment derived from the proteolysis of C3b, have revealed a domain configuration, including six macroglobulin domains (MG1-MG6; nomenclature follows ref. 5) arranged in a ring, termed the beta-ring. However, because neither C3 nor C3c is active in complement activation and regulation, questions about function can be answered only through direct observations on C3b. Here we present a structure of C3b that reveals a marked loss of secondary structure in the CUB (for 'complement C1r/C1s, Uegf, Bmp1') domain, which together with the resulting translocation of the thioester domain provides a molecular basis for conformational changes accompanying the conversion of C3 to C3b. The total conformational changes make many proposed ligand-binding sites more accessible and create a cavity that shields target peptide bonds from access by factor I. A covalently bound N-acetyl-l-threonine residue demonstrates the geometry of C3b attachment to surface hydroxyl groups.