Structure of Ca(2+)-loaded human grancalcin.

Grancalcin is a cytosolic Ca(2+)-binding protein originally identified in human neutrophils. It belongs to a new class of EF-hand proteins, called PEF proteins, which contain five EF-hand motifs. At the N-terminus of grancalcin there is a approximately 50 residue-long segment rich in glycines and prolines. The fifth EF-hand, unpaired within the monomer, provides a means for dimerization through pairing with its counterpart in a second molecule. The structure of full-length grancalcin in the apo form and with one EF3 within the dimer occupied by a Ca(2+) ion have been determined. Although the N-terminal segment was present in the molecule, this part was disordered in the crystals. Here, the structure of a truncated form of grancalcin, which is lacking 52 N-terminal residues, in the presence and absence of Ca(2+) is presented. In the Ca(2+)-bound form the ions are found in the EF1 and EF3 hands. Binding of Ca(2+) to these two EF hands produces only minor conformational changes, mostly within the EF1 Ca(2+)-binding loop. This observation supports the hypothesis, formulated on the basis of the structure of a homologous protein ALG-2 which shows significant differences in the orientation of EF4 and EF5 compared with grancalcin, that calcium is a necessary factor but not sufficient alone for inducing a significant conformational change in PEF proteins.

Biochemical characterization of the penta-EF-hand protein grancalcin and identification of L-plastin as a binding partner.

Grancalcin is a recently described Ca(2+)-binding protein especially abundant in human neutrophils. Grancalcin belongs to the penta-EF-hand subfamily of EF-hand proteins, which also comprises calpain, sorcin, peflin, and ALG-2. Penta-EF-hand members are typified by two novel types of EF-hands: one that binds Ca(2+) although it has an unusual Ca(2+) coordination loop and one that does not bind Ca(2+) but is directly involved in homodimerization. We have developed a novel method for purification of native grancalcin and found that the N terminus of wild-type grancalcin is acetylated. This posttranslational modification does not affect the secondary structure or conformation of the protein. We found that both native and recombinant grancalcin always exists as a homodimer, regardless of the Ca(2+) load. Flow dialysis showed that recombinant grancalcin binds two Ca(2+) per subunit with positive cooperativity and moderate affinity ([Ca(2+)](0.5) of 25 and 83 microm in the presence and absence of octyl glycoside, respectively) and that the sites are of the Ca(2+)-specific type. Furthermore, we showed, by several independent methods, that grancalcin undergoes important conformational changes upon binding of Ca(2+) and subsequently exposes hydrophobic amino acid residues, which direct the protein to hydrophobic surfaces. By affinity chromatography of solubilized human neutrophils on immobilized grancalcin, L-plastin, a leukocyte-specific actin-bundling protein, was found to interact with grancalcin in a negative Ca(2+)-dependent manner. This was substantiated by co-immunoprecipitation of grancalcin by anti-L-plastin antibodies and vice versa.

Crystal structure of human grancalcin, a member of the penta-EF-hand protein family.

Grancalcin is a Ca(2+)-binding protein expressed at high level in neutrophils. It belongs to the PEF family, proteins containing five EF-hand motifs and which are known to associate with membranes in Ca(2+)-dependent manner. Prototypic members of this family are Ca(2+)-binding domains of calpain. Our recent finding that grancalcin interacts with L-plastin, a protein known to have actin bundling activity, suggests that grancalcin may play a role in regulation of adherence and migration of neutrophils. The structure of human grancalcin has been determined at 1.9 A resolution in the absence of calcium (R-factor of 0.212 and R-free of 0.249) and at 2. 5 A resolution in the presence of calcium (R-factor of 0.226 and R-free of 0.281). The molecule is predominantly alpha-helical: it contains eight alpha-helices and only two short stretches of two-stranded beta-sheets between the loops of paired EF-hands. Grancalcin forms dimers through the association of the unpaired EF5 hands in a manner similar to that observed in calpain, confirming this mode of association as a paradigm for the PEF family. Only one Ca(2+) was found per dimer under crystallization conditions that included CaCl(2). This cation binds to EF3 in one molecule, while this site in the second molecule of the dimer is unoccupied. This unoccupied site shows higher mobility. The structure determined in the presence of calcium, although does not represent a fully Ca(2+)-loaded form, suggests that calcium induces rather small conformational rearrangements. Comparison with calpain suggests further that the relatively small magnitude of conformational changes invoked by calcium alone may be a characteristic feature of the PEF family. Moreover, the largest differences are localized to the EF1, thus supporting the notion that calcium signaling occurs through this portion of the molecule and that it may involve the N-terminal Gly/Pro rich segment. Electrostatic potential distribution shows significant differences between grancalcin and calpain domain VI demonstrating their distinct character.

Crystallization and preliminary X-ray analysis of human grancalcin, a novel cytosolic Ca2+-binding protein present in leukocytes.

Recombinant human grancalcin, a calcium-binding protein from leukocytes, has been crystallized in the presence or absence of Ca(2+) by the vapor-diffusion method. Two crystal forms of apo grancalcin were obtained: space group P2(1), with unit-cell parameters a = 48.4, b = 81.1, c = 46.6 A, beta = 111.3 degrees, diffracting to 1.9 A, and space group C2, with unit-cell parameters a = 97.0, b = 51.9, c = 75.9 A, beta = 108.5 degrees, diffracting to 2.4 A. Crystals were also grown in the presence of 5 mM Ca(2+). They also belong to space group C2, with unit-cell parameters a = 97.4, b = 50.3, c = 77.6 A, beta = 108.2 degrees, which are very similar to the second apo grancalcin form. These crystals diffract to 2.5 A.

Membrane capacitance techniques to monitor granule exocytosis in neutrophils.

Cell membranes behave like electrical capacitors and changes in cell capacitance therefore reflect changes in the cell area. Monitoring capacitance can thus be used to study dynamic cellular phenomenon involving rapid changes in cell surface, such as exo- and/or endocytosis. In this review focus is on the use of capacitance techniques to study exocytosis in human neutrophils. We compare the whole-cell and the cell-attached capacitance techniques, and we review the complete literature dealing with capacitance measurements in human neutrophils.

Timing, targeting and sorting of azurophil granule proteins in human myeloid cells.

Biosynthesis of 3 human granule proteins, myeloperoxidase, defensin and lysozyme, all present in azurophil granules, was investigated in normal bone marrow cells and in the promyelocytic cell line HL-60 to see whether differences in timing of biosynthesis could explain the well established differences in their subcellular localization in the mature neutrophil (targeting), and whether differences exist in the efficiencies by which granule proteins are retained in cells (sorting). Normal human bone marrow cells were separated into three bands by density gradient centrifugation. Band 1 contains band and segmented cells, band 2 mainly myelocytes, metamyelocytes and some band cells, and band 3 myeloblasts and promyelocytes in addition to megakaryocytes and proerythroblasts. Cells from these bands, as well as undifferentiated HL-60 cells, were pulsed with radiolabeled cysteine and methionine, and biosynthesis of granule proteins was subsequently evaluated by immunoprecipitation and quantified by phosphorimaging. Myeloperoxidase synthesis was maximal in cells from band 3 while defensin biosynthesis was maximal in cells from band 2. Lysozyme was synthesized in cells from all bands but was maximal in cells from band 2. These results are in agreement with our hypothesis that timing of biosynthesis determines the localization of individual granule proteins. While myeloperoxidase and defensins were efficiently retained in immature cells (band 3), a significant fraction of lysozyme was routed out of the cells, showing that differences exist in the sorting of granule proteins between constitutive and regulated secretion. In addition, defensin was less efficiently retained in cells from band 2 than from band 3, indicating that sorting mechanisms may depend on the stage of cell maturation.

Capacitance flickers and pseudoflickers of small granules, measured in the cell-attached configuration.

We have studied exocytosis of single small granules from human neutrophils by capacitance recordings in the cell-attached configuration. We found that 2.2% of the exocytotic events were flickers. The flickers always ended with a downward step. This indicates closing of the fusion pore. During flickering, the fusion pore conductance remained below 1 nS, and no net membrane transfer was detectable. After fusion pore expansion beyond 1 nS the pore expanded irreversibly, leading to rapid full incorporation of the granule/vesicle into the plasma membrane. Following exocytosis of single granules, a capacitance decrease directly related to the preceding increase was observed in 7% of the exocytotic events. This decrease followed immediately after irreversible pore expansion, and is presumably triggered by full incorporation of the vesicle into the patch membrane. The capacitance decrease could be interpreted as endocytosis triggered by exocytosis. However, the gradual decrease could also reflect a decrease in the "free" patch area following incorporation of an exocytosed vesicle. We conclude that non-stepwise capacitance changes must be interpreted with caution, since a number of factors go into determining cell or patch admittance.

Granules and secretory vesicles in human neonatal neutrophils.

Neonates have an increased susceptibility to bacterial infections that may be due to defective adherence and migration of neonatal neutrophils (NN). Because receptors of relevance for these activities are located mainly in intracellular granules and secretory vesicles that have only recently been characterized in adult neutrophils (AN), we investigated whether the same structures are present in NN and to what extent they are mobilized in response to chemotactic and inflammatory mediators. Subcellular fractionation of NN on a three-layer Percoll density gradient revealed that secretory vesicles, identified by latent alkaline phosphatase and albumin, are present in NN. We also demonstrated the presence of gelatinase granules distinct from specific granules, although the content of their respective markers, gelatinase and lactoferrin, was markedly reduced. The low content of lactoferrin may explain an observed lower isopycnic density of specific granules in NN. Mobilization of granules by a variety of stimuli was slightly higher in NN compared with AN, whereas mobilization of secretory vesicles was normal. This shows that NN contain both secretory vesicles and all subsets of granules identified in AN, and that these are readily mobilized, although a marked structural difference exists between peroxidase-negative granules of NN and AN that may reflect differences during myelopoiesis.

An ELISA for grancalcin, a novel cytosolic calcium-binding protein present in leukocytes.

Grancalcin is a newly discovered cytosolic calcium-binding protein, belonging to the group of EF-hand proteins. Grancalcin is specifically associated with cells originating in the bone marrow. Grancalcin binds reversibly to secretory vesicles and plasma membranes in human neutrophils and might therefore play a role in the regulation of vesicle/granule exocytosis. We describe here the production of recombinant grancalcin, the generation of antibodies to the protein and the development of a specific, accurate and sensitive ELISA for the detection of human leukocytic grancalcin. This ELISA may be useful for monitoring leukocyte infiltration into tissues.

The exocytotic fusion pore of small granules has a conductance similar to an ion channel.

We measured capacitance changes in cell attached patches of human neutrophils using a high frequency lock-in method. With this technique the noise level is reduced to 0.025 fF such that capacitance steps of 0.1 fF are clearly detected corresponding to exo- and endocytosis of single 60 nm vesicles. It is thus possible to detect almost all known exocytotic and endocytotic processes including exocytosis of small neurotransmitter containing vesicles in most cell types as well as endocytosis of coated and uncoated pits. In neutrophils we demonstrate a stepwise capacitance decrease generated by 60-165 nm vesicles as expected for endocytosis of coated and non-coated pits. Following ionomycin stimulation a stepwise capacitance increase is observed consisting of 0.1-5 fF steps corresponding to the different granule types of human neutrophils from secretory vesicles to azurophil granules. The opening of individual fusion pores is resolved during exocytosis of 200 nm vesicles. The initial conductance has a mean value of 150 pS and can be as low as 35 pS which is similar to the conductance of many ion channels suggesting that the initial fusion pore is formed by a protein complex.

Mobilization of granules and secretory vesicles during in vivo exudation of human neutrophils.

The extent of mobilization of four different intracellular compartments was measured during in vivo exudation of neutrophils into skin chambers and compared with resting neutrophils obtained from blood. Exudation of neutrophils induced increased surface expression of alkaline phosphatase, complement receptor 1, and Mac-1, and a complete loss of L-selectin. The increase in the content of surface molecules in the plasma membrane is in accordance with complete mobilization of secretory vesicles. Granule matrix proteins were secreted into the chamber fluid by the exudated neutrophils and the exocytosed proteins were recovered in the skin chamber fluid. Release of gelatinase from gelatinase granules was 38.1%, lactoferrin release from specific granules was 21.9%, and myeloperoxidase release from azurophil granules was 7.0%, clearly illustrating a hierarchy in mobilization among granules. When exudate neutrophils were stimulated with FMLP, additional mobilization of granules was observed and the rank order regarding release was preserved. This is the first report to evaluate the mobilization of secretory vesicles during in vivo exudation of human neutrophils. It is shown that secretory vesicles are regulated exocytotic vesicles that are fully mobilized during in vivo exudation. Once exocytosed, secretory vesicles are not re-formed within a period of 6 h.

Lysozyme in human neutrophils and plasma. A parameter of myelopoietic activity.

Lysozyme was found to be present in all three types of human neutrophil granules (azurophil-, specific- and gelatinase granules) as determined by subcellular fractionation, employing a three-layer Percoll gradient and measurement of lysozyme by a novel ELISA. The content of lysozyme was also measured in plasma. In contrast to other neutrophil granule proteins (lactoferrin, NGAL, and gelatinase), plasma lysozyme was unaffected by increase in the number of circulating neutrophils induced by intravenous administration of methylprednisolone to healthy individuals. Also in contrast to lactoferrin, NGAL, and gelatinase, plasma lysozyme was found to rise 1 week prior to detectable increases in the number of circulating neutrophils in patients undergoing allogeneic bone marrow transplantation. We conclude that plasma lysozyme is a parameter of myelopoietic activity and may be useful as a marker for bone marrow repopulation after transplantation.

Purification of lysozyme from human neutrophils, and development of an ELISA for quantification in cells and plasma.

Lysozyme was purified from exocytosed granule material from PMA-stimulated human neutrophils by polyethyleneglycol precipitation, cation exchange chromatography and molecular sieve chromatography. Rabbit antibodies were biotinylated and affinity purified on a lysozyme column, for subsequent development of a novel ELISA. This ELISA for lysozyme is sensitive and accurate, and applicable to determination of lysozyme in neutrophils and plasma.

[Ultrasound and lymphography in malignant lymphoma. Comparison with consideration to staging]. / Ultralyd og lymfografi ved malignt lymfom. Sammenligning med henblik på stadieinddeling.

The diagnostic value of abdominal ultrasound and lymphography was compared in the staging of 88 consecutive patients with malignant lymphoma, examined during the period March 1990 to April 1991. Lymphography was used as the reference method in the evaluation of the paraaortic and iliac lymphnodes. In 19% of the patients ultrasound examination could not be accomplished optimally, and these results were evaluated in a separate group. Lymphography demonstrated involvement of retroperitoneal lymphnodes in 27 patients, among these ultrasound was false negative in seven (= 26%). In ten patients ultrasound examination demonstrated lymphoma outside the lymphographic area. No false positive ultrasound examinations were found in the group with negative lymphography. Ultrasound cannot replace lymphography, but is an important supplement, and in those cases where ultrasound reveals lymphomas in the lymphographic area, lymphography can be omitted as ultrasound has a high predictive value.

Isolation and characterization of gelatinase granules from human neutrophils.

We recently confirmed the existence of gelatinase granules as a subpopulation of peroxidase-negative granules by double-labeling immunogold electron microscopy on intact cells and by subcellular fractionation. Further characterization of gelatinase granules has been hampered by poor separation of specific and gelatinase granules on both two-layer Percoll gradients and sucrose gradients. We have developed a three-layer Percoll density gradient that allows separation of the different granules and vesicles from human neutrophils; in particular, it allows separation of specific and gelatinase granules. This allows us to characterize these two granule populations with regard to their content of membrane proteins, which become incorporated into the plasma membrane during exocytosis. We found that gelatinase granules, defined as peroxidase-negative granules containing gelatinase but lacking lactoferrin, contain 50% of total cell gelatinase, with the remaining residing in specific granules. Furthermore, we found that 20% to 25% of both the adhesion protein Mac-1 and the NADPH-oxidase component cytochrome b558 is localized in gelatinase granules. Although no qualitative difference was observed between specific granules and gelatinase granules with respect to cytochrome b558 and Mac-1, stimulation of the neutrophil with FMLP resulted in a selective mobilization of the least dense peroxidase-negative granules, ie, gelatinase granules, which, in concert with secretory vesicles, furnish the plasma membrane with Mac-1 and cytochrome b558. This shows that gelatinase granules are functionally important relative to specific granules in mediating early inflammatory responses.

Human neutrophil granules and secretory vesicles.

The traditional classification of neutrophil granules as peroxidase-positive (azurophil, or primary) and peroxidase-negative (specific or secondary) has proven to be too simple to explain the differential exocytosis of granule proteins and incorporation of granule membrane into the plasma membrane which is an important aspect of neutrophil activation. Combined subcellular fractionation and immunoelectron microscopy has revealed heterogeneity among both peroxidase-positive and peroxidase-negative granules with regard to their content, mobilization and time of formation. Peroxidase-negative granules may be classified according to their content of lactoferrin and gelatinase: 15% of peroxidase-negative granules contain lactoferrin, but no gelatinase. 60% contain both lactoferrin and gelatinase. The term specific or secondary granule should be reserved for these two subsets. In addition, 25% of peroxidase-negative granules contain gelatinase but no lactoferrin. These should be termed gelatinase granules or tertiary granules. Gelatinase granules are formed later than specific granules and mobilized more readily. In addition, a distinct, highly mobilizable intracellular compartment, the secretory vesicle, has now been recognized as an important store of surface membrane-bound receptors. This compartment is formed in band cells and segmented cells by endocytosis. This heterogeneity among the neutrophil granules is of functional significance, and may also be reflected in the dysmaturation which is an important feature of myeloproliferative and myelodysplastic disorders.

Ca(2+)-dependent translocation of cytosolic proteins to isolated granule subpopulations and plasma membrane from human neutrophils.

In order to identify cytosolic proteins involved in control of granule exocytosis in human neutrophils, subcellular fractions enriched in each of the 3 major granule subsets were incubated with cytosol from neutrophils in the presence or absence of Ca2+. After washing, proteins were eluted from the organelles by EGTA. Annexins I, II, IV and VI were found to bind to all organelles studied. In addition, a 28-kDa protein was found to bind exclusively to plasma membranes and secretory vesicles, the most readily exocytosed organelle of neutrophils. Ca(2+)-dependent association of cytosolic proteins to different granule subsets may control differential exocytosis of granules.

Ca2+ and vasopressin release in isolated rat neurohypophysis: differential effects of four classes of Ca2+ channel ligands.

In order to study the role of different types of voltage-sensitive Ca2+ channels (VSCC) in stimulus-secretion coupling in peptidergic neurons, effects of 4 major classes of pharmacological agents have been examined on evoked vasopressin release from isolated rat neurohypophyses. omega-Conotoxin GVIA (omega-CgTX), a potent blocker of N- and L-type Ca2+ channels, inhibited vasopressin release evoked electrically as well as by high K+. With maximal inhibition, release was decreased to 50% and 75% of control for electrical and 100 mM K+ stimulation, respectively. This stimulation mode-related difference in release sensitivity to omega-CgTX paralleled its stimulation mode-related sensitivity to tetrodotoxin, suggesting that the omega-CgTX-sensitive Ca2+ entry played a larger role when release was activated by action potentials invading nerve terminals. These data, and the characteristics of [125I]omega-CgTX binding to plasma membranes from bovine neurohypophyses, are consistent with N-type Ca2+ channels being responsible for the omega-CgTX-sensitive component of vasopressin release. Verapamil and diltiazem (phenylalkylamine and benzothiazepine, respectively) inhibited secretion in a pattern suggesting non-identical sets of action sites, and in a manner partly additive with inhibition by omega-CgTX. This inhibition by verapamil and diltiazem appeared at least in part to involve sites different from L channels. Several dihydropyridines known to act as agonists or antagonists at L channels did not affect vasopressin release (evoked either electrically or by high K+) in a specific manner. A significant component of neuropeptide release may depend on Ca2+ entry through omega-CgTX- and dihydropyridine-insensitive routes.