Roles of individual kringle domains in the functioning of positive and negative effectors of human plasminogen activation

In order to identify the individual contributions of the kringle (K) domains of human plasminogen (Pg) to the epsilon-aminocaproic acid (EACA) induced stimulation of Pg activation by low-molecular-weight urokinase-type plasminogen activator (LMW-uPA) and inhibition of this same activation by Cl-, we constructed the most conservative recombinant- (r-) Pg mutants possible that would greatly reduce the strength of the EACA binding site in the omega-amino acid binding kringles, [K1Pg] ([D139-->N]r-Pg), [K4Pg] ([D413-->N]r-Pg), and [K5Pg] ([D515--N]r-Pg). In each case, this involved mutation of a critical Asp (to Asn) within these three kringle domains in intact Pg. The three r-mutants were expressed in r-baculovirus-infected lepidopteran insect (Trichoplusia ni) cells. In the presence of Cl-, the positive activation effector, EACA, first stimulated and then inhibited the LMW-uPA-catalyzed initial activation of wild-type (wt) r-[Glu1]Pg and, to a lesser extent, the [K5Pg] mutant, [D518-->N/Glu1]r-Pg. The concentration of EACA that produced 50% stimulation of activation (C50) occurred at 3.3 mM for wtr-[Glu1]Pg and at 0.7 mM for [D518-->N/Glu1]r-Pg. Subsequent inhibition by EACA occurred with a C50 of approximately 15 mM and is likely due to inhibition of the amidolytic activity of plasmin generated during the activation. Similar initial activation rates of both [D139-->N]r-Pg and [D413N]r-Pg did not display this initial EACA-mediated stimulatory phase but did undergo ultimate inhibition with a C50 for this process that was similar to wtr-[Glu1]Pg and [D518-->N/Glu1]r-Pg.(ABSTRACT TRUNCATED AT 250 WORDS)     

Modification of the calcium and calmodulin sensitivity of the type I adenylyl cyclase by mutagenesis of its calmodulin binding domain

The type I adenylyl cyclase is directly stimulated by Ca2+ and calmodulin in vitro, and the enzyme is also stimulated by increases in intracellular Ca2+ in vivo. Ca2+ stimulation of the enzyme in vivo may be due to direct interactions of the enzyme with Ca2+ and calmodulin or to an indirect mechanism involving stimulation of the enzyme by Ca(2+)-activated protein kinases. In this study, we have made several point mutations within the calmodulin binding domain to determine if the Ca2+ sensitivity of the enzyme can be modified by mutagenesis. The catalytic activities of the mutant enzymes were comparable to wild type type I adenylyl cyclase. Substitution of Cys-507 with Ser-507 did not have significant effects on the calmodulin or Ca2+ sensitivity of the enzyme. However, replacement of Lys-504 with Asp caused a 4-fold decrease in sensitivity to Ca2+. Ca2+ and calmodulin stimulation were abolished by substitution of Phe-503 with Arg-503. Stimulation of type I adenylyl cyclase activity in vivo by intracellular Ca2+ was also greatly diminished with the Arg-503 mutant indicating that Ca2+ stimulation of the enzyme in vivo is due primarily to direct interactions with calmodulin and Ca2+. These data demonstrate that the Ca2+ sensitivity of this enzyme can be modulated by point mutagenesis within the putative calmodulin binding domain and indicate that the enzyme can be directly regulated by Ca2+ and calmodulin in vivo.     

Expression of annexin I, II, V, and VI by rat osteoblasts in primary culture: stimulation of annexin I expression by dexamethasone

To determine whether rat osteoblasts synthesize proteins of the annexin family and to evaluate the extent to which glucocorticoids modulate the expression of annexins by these cells, osteoblasts were grown in primary cultures in the absence or presence of dexamethasone, and the expression of annexins was evaluated by immunoblotting using polyclonal antibodies against human annexins. Four different annexins (I, II, V, and VI) were found to be expressed by rat osteoblasts. The expression of annexin I, but not the other annexins studied, was increased in osteoblasts cultured in the presence of dexamethasone (173 +/- 33% increase comparing untreated cells and cells treated for 10 days with 5 x 10(-7) M dexamethasone). Increased expression of annexin I was observed after the third day of exposure to dexamethasone and rose thereafter until day 10; annexin I expression increased with dexamethasone concentrations above 10(-10) M throughout the range of concentrations studied. The increase in annexin I protein was associated with an increase in annexin I mRNA and was completely blocked by the concomitant addition of the glucocorticoid receptor antagonist RU 38486. The increase in annexin I content following dexamethasone treatment was associated with an increase in alkaline phosphatase activity and PTH-induced cAMP stimulation, whereas phospholipase A2 activity in the culture medium was reduced to undetectable levels. The finding that four annexins are expressed in rat osteoblasts in primary culture raises the possibility that these proteins could play an important role in bone formation by virtue of their ability to bind calcium and phospholipids, serve as Ca2+ channels, interact with cytoskeletal elements, and/or regulate phospholipase A2 activity. In addition, the dexamethasone-induced increase in annexin I may represent a mechanism by which glucocorticoids modify osteoblast function.     

Effects of profilin-annexin I association on some properties of both profilin and annexin I: modification of the inhibitory activity of profilin on actin polymerization and inhibition of the self-association of annexin I and its interactions with liposomes

We have previously shown that annexin I, a member of a family of calcium-dependent phospholipid and membrane binding proteins, interacts with profilin with high specificity and affinity. This finding further suggests that annexin I is involved through profilin in the regulation of membrane-cytoskeleton organization. We have investigated the consequences of a complex formed by these two proteins on the functions of both profilin and annexin I. Annexin I is able to modify the inhibitory effect of profilin on actin polymerization. This action is partial and the mechanism involved appears to be complex. On the other hand, the association between annexin I and profilin is sufficiently strong to inhibit the self-association of annexin I. The binding capacity of annexin I to liposomes containing phosphatidylserine, which mimics annexin I binding to membranes, is also decreased by profilin. This binding is nevertheless restored when phosphatidylinositol 4,5-biphosphate (PtdInsP2) is included in the liposomes. Finally, the capacity of annexin I to aggregate liposomes is also modified. It is worthwhile mentioning that the liposomes-binding and liposomes-aggregating activities of annexin I are independently regulated. The cell localization and functions of annexin I and profilin suggest that interaction between these two proteins may be directly implicated in the regulation of membrane-cytoskeleton. The phospholipid composition of membranes may be one of the modulating factors.     

Identification and mutagenesis of a highly conserved domain in troponin T responsible for troponin I binding: potential role for coiled coil interaction

Troponin T (TnT), a thin filament myofibrillar protein, is essential for the Ca2+ regulation of striated muscle contraction in vertebrates, both in vivo and in vitro. To understand the role of TnT in this process, its interaction with two other troponin components, troponin I (TnI) and troponin C (TnC) was examined by using the yeast two hybrid system, which is a genetic approach to detect protein-protein interactions. Computer assisted analysis of phylogenetically distant TnT amino acid sequences unveiled a highly conserved protein domain that is characterized by a heptad repeat (HR) motif with a potential for alpha-helical coiled coil formation. A similar, potentially coiled coil forming domain is also conserved in all known TnI sequences. These protein motifs appeared to be the regions where TnI-TnT interaction may take place. Deletions and point mutations in TnT, which disrupted its HR motif, severely reduced or abolished TnI binding, but binding to TnC was not affected, indicating that the TnT-TnI and TnT-TnC binary interactions can be uncoupled. Remarkably, the truncated fragments of TnT and TnI in which the HR motifs were retained showed binary interaction in the yeast two hybrid system. It was also observed that the formation of the TnT-TnI heterodimers is favored over the homodimers TnT-TnT and TnI-TnI. These results indicate that the evolutionarily conserved HR motifs may play a role in TnT-TnI dimerization, presumably through the formation of alpha-helical coiled coils.     

Molecular dynamics of annexin I in normal and infected cells

Annexins are a family of proteins present within the tissues of all multicellular organisms, mammalian erythrocytes excepted. The property shared by all annexins is the calcium and membrane binding. Annexins are constituted of two domains. The N-terminal domain gives the molecule its specificity and the C-terminal domain, highly conserved, containing 4 repetitions of 70 amino-acids, gives the common properties. Although numerous important works were performed, the exact function of annexins is not unraveled. They participate to many cellular processes as for instance exocytosis, endocytosis or phagosome maturation. Many hypotheses, supported by experimental results, have been proposed. In this review, we propose a summary of the principal characteristics of annexins and we discuss the main hypotheses proposed for their functions.     

Site-directed mutagenesis of the human A1 adenosine receptor: influences of acidic and hydroxy residues in the first four transmembrane domains on ligand binding

To provide new insights into ligand/A1 adenosine receptor (A1 AR) interactions, site-directed mutagenesis was used to test the role of several residues in the first four transmembrane (TM) domains of the human A1 AR. Based on multiple sequence analysis of all known ARs, both acidic (glutamic acid and aspartic acid) and polar hydroxy (serine and threonine) amino acids were identified that could potentially play a role in binding adenosine. Glu16 (TM1), Asp55 (TM2), Ser93 and Ser94 (TM3), Ser135 (TM4), and Thr 141 (TM4) were identified in all ARs, and Ser6 and Ser23 (TM1) were identified in all A1 ARs. To test the role of these residues, each was individually mutated to alanine. When Ala6, Ala23, Ala50, Ala93, Ala135, and Ala141 constructs were tested, affinities for [3H]2-chloro-N6-cyclopentyladenosine (CCPA) and [3H]1,3-dipropyl-8-cyclopentylxanthine (DPCPX) were similar to those seen for the wild-type receptor. After conversion of Glu16 to Ala16, the affinity for [3H]CCPA and other agonists fell 10-100-fold, whereas the affinity for [3H]DPCPX and other antagonists was not affected. After conversion of Asp55 to Ala55, the affinity for [3H]CCPA and other agonists increased < or = 100-fold, whereas the affinity for [3H]DPCPX and other antagonists was not affected. Studies of the Ala55 construct also revealed that Asp55 is responsible for allosteric regulation of binding by sodium because the affinity for [3H]CCPA did not change over broad ranges of sodium concentrations. When Ser94 was converted to Ala94, A1 AR immunoreactivity was present on stable cell lines; however, functional binding sites could not be detected. When Ser94 was converted to Thr94, the affinity for some xanthine antagonists fell. These data show that Glu16 in TM1 and Asp55 in TM2 play important roles in agonist/A1 AR interactions and show that Asp55 is responsible for allosteric regulation of ligand/A1 AR binding by sodium. We also identify Ser94 as an important site for ligand binding.     

Cleavage of annexin I in human neutrophils is mediated by a membrane-localized metalloprotease

A truncated form of annexin I, formed during Ca2+-induced translocation to neutrophil specific granules and secretory vesicles/plasma membranes, is generated through the action of an endogenous membrane protease. The cleavage of annexin I is inhibited by the metalloprotease inhibitor 1,10-phenanthroline as well as by Triton X-100 and dithiothreitol, classifying the protease as a membrane-bound, thiol-dependent metalloprotease. The cleavage site is located close to the N-terminal of annexin I, leaving a truncated form of the molecule, des1-8 annexin I, that contains the Ca2+-binding sites, as well as a number of phosphorylation sites of importance for the function of the protein. When assessing binding capacity to different neutrophil organelles, full-length annexin I bound to azurophil granules, specific granules, and secretory vesicles/plasma membranes, while des1-8 annexin I only bound to specific granules and secretory vesicles/plasma membranes, but not to azurophil granules (C. Sj?lin, C. Dahlgren, Biochim. Biophys. Acta 1281 (1996) 227-234). This implies that there are different mechanisms of binding to neutrophil organelles of full-length annexin I and the truncated form, and that cleavage of annexin I might be of regulatory importance for the degranulation process.     

Ezrin contains cytoskeleton and membrane binding domains accounting for its proposed role as a membrane-cytoskeletal linker

Ezrin, a widespread protein present in actin-containing cell-surface structures, is a substrate of some protein tyrosine kinases. Based on its primary and secondary structure similarities with talin and band 4.1 it has been suggested that this protein could play a role in linking the cytoskeleton to the plasma membrane (Gould, K.L., A. Bretscher, F.S. Esch, and T. Hunter. 1989. EMBO (Eur. Mol. Biol. Organ.), J. 8:4133-4142; Turunen, O., R. Winqvist, R. Pakkanen, K.-H. Grzeschik, T. Wahlstr?m, and A. Vaheri. 1989. J. Biol. Chem. 264:16727-16732). To test this hypothesis, we transiently expressed the complete human ezrin cDNA, or truncated cDNAs encoding the amino- and carboxy-terminal domains of the protein, in CV-1 cells. Protein epitope tagging was used to unambiguously determine the subcellular distribution of the protein encoded by the transfected cDNA. We show that this protein is concentrated underneath the dorsal plasma membrane in all actin-containing structures and is partially detergent insoluble. The amino-terminal domain displays the same localization but is readily extractable by nonionic detergent. The carboxy-terminal domain colocalizes with microvillar actin filaments as well as with stress fibers and remains associated with actin filaments after detergent extraction, and with disorganized actin structures after cytochalasin D treatment. Our results clearly demonstrate that ezrin interacts with membrane-associated components via its amino-terminal domain, and with the cytoskeleton via its carboxy-terminal domain. The amino-terminal domain could include the main determinant that restricts the entire protein to the cortical cytoskeleton in contact with the dorsal plasma membrane and its specialized microdomains such as microvilli, microspikes and lamellipodia.     

Interaction of chemokine receptor CCR5 with its ligands: multiple domains for HIV-1 gp120 binding and a single domain for chemokine binding

CCR5 is a chemokine receptor expressed by T cells and macrophages, which also functions as the principal coreceptor for macrophage (M)-tropic strains of HIV-1. To understand the molecular basis of the binding of chemokines and HIV-1 to CCR5, we developed a number of mAbs that inhibit the various interactions of CCR5, and mapped the binding sites of these mAbs using a panel of CCR5/CCR2b chimeras. One mAb termed 2D7 completely blocked the binding and chemotaxis of the three natural chemokine ligands of CCR5, RANTES (regulated on activation normal T cell expressed and secreted), macrophage inflammatory protein (MIP)-1alpha, and MIP-1beta, to CCR5 transfectants. This mAb was a genuine antagonist of CCR5, since it failed to stimulate an increase in intracellular calcium concentration in the CCR5 transfectants, but blocked calcium responses elicited by RANTES, MIP-1alpha, or MIP-1beta. This mAb inhibited most of the RANTES and MIP-1alpha chemotactic responses of activated T cells, but not of monocytes, suggesting differential usage of chemokine receptors by these two cell types. The 2D7 binding site mapped to the second extracellular loop of CCR5, whereas a group of mAbs that failed to block chemokine binding all mapped to the NH2-terminal region of CCR5. Efficient inhibition of an M-tropic HIV-1-derived envelope glycoprotein gp120 binding to CCR5 could be achieved with mAbs recognizing either the second extracellular loop or the NH2-terminal region, although the former showed superior inhibition. Additionally, 2D7 efficiently blocked the infectivity of several M-tropic and dual-tropic HIV-1 strains in vitro. These results suggest a complicated pattern of HIV-1 gp120 binding to different regions of CCR5, but a relatively simple pattern for chemokine binding. We conclude that the second extracellular loop of CCR5 is an ideal target site for the development of inhibitors of either chemokine or HIV-1 binding to CCR5.     

Structure of human annexin I: comparison of homology modelling and crystallographic experiment

A model of domain II of annexin I has been built by homology modelling using an annexin V crystal structure as a template. The method used is based on that of Summers and Karplus (J Mol Biol (1989) 210, 785-811) and involves the calculation of torsion-angle rotational energy maps to position side chains. The RMS deviation of the backbone heavy atoms between the model and a crystal structure of annexin I is 1.1 A. Similarities and differences in the experimental and model-derived side-chain rotameric conformations and hydrogen-bonding interactions are examined. It is found that whereas many of the side chains are well positioned some of those placed using the 'entropy argument' in which the broadest of the available minima are preferred, are erroneous. The domain is subjected to molecular dynamics simulation in explicit solvent. The simulations are found to 'correct' some of the side-chain rotamer positions that were poorly placed in the homology modelling. Considerable helix instability is seen in the simulations, consistent with the requirement of domain interactions for the structural integrity of the protein.     

Developmental profile of a caltrin-like protease inhibitor, P12, in mouse seminal vesicle and characterization of its binding sites on sperm surface

We examined the developmental profile of a kazal-type trypsin inhibitor (P12) of Mr 6126 in mouse seminal vesicle, characterized its binding sites on the surface of sperm, and assessed its effect on Ca2+ uptake by spermatozoa. Among the genital tracts of adult mice, P12 was found only in the male accessory glands including seminal vesicle, coagulating gland, and prostate. It was immunolocalized on the luminal epithelium of the primary and secondary folds in both the seminal vesicle and coagulating gland, and on the folds projecting into the lumen of the glandular alveolus in the prostate. The protein and its RNA message in seminal vesicle did not appear in the prepubertal period, but expression coincided with maturation. Castration of adult mice resulted in cessation of P12 expression. Treatment of the castrated mice with testosterone propionate in corn oil restored the protein expression in the seminal vesicle. Spermatozoa collected from caudal epididymis were devoid of P12. Cytochemical study illustrated a P12-binding region on the anterior acrosomes of cells preincubated with P12. Analysis of equilibrium data from the binding assay using 125I-P12 with a Scatchard plot showed a single type of P12-binding sites on sperm, with an apparent dissociation constant of 70.15 +/- 5.25 nM and the capacity of 1.49 +/- 0.06 x 10(6) binding sites/cell. The protein could serve as a calcium transport inhibitor to suppress a great extent of Ca2+ uptake by spermatozoa. The immunohistochemical staining patterns of testis revealed that the P12-binding sites appeared on postmeiotic cells such as spermatids and spermatozoa, but were absent in Leydig cells, Sertoli cells, spermatogonia, and spermatocytes in seminiferous tubules.     

Replacement of terminal cysteine with histidine in the metallothionein alpha and beta domains maintains its binding capacity

To generate novel forms of metal-binding proteins, six mutant mouse metallothionein (MT) 1 fragments, in which a terminal cysteine residue was replaced by histidine, were expressed in Escherichia coli. The spectroscopic and analytical results showed that the alphaMT (C33H, C36H, C41H, C57H) and betaMT (C5H, C13H) mutant forms bound 4 and 3 Zn(II) atoms per molecule of protein to the nearest integer, even though in C41H and C5H, species of lower stoichiometry were also detected. In Cd(II) titrations, all the Zn(II) ions bound to the mutant proteins were displaced from the binding sites, giving rise to Cd-mutated MT forms with 4 and 3 Cd(II), respectively. However, although Cys-to-His substitutions maintained the binding capacity of the MT fragments, they caused structural changes with respect to the wild-type proteins. While C13H, C36H and C57H seem to contain Zn(II)-aggregates that are closely related to those of the wild-type proteins, only C41H and C57H gave rise to Cd(II)-aggregates similar to those of Cd4-alphaMT, where the His residue plays the role of the substituted Cys. Despite the structural implications of the Cys-to-His replacement, the dissociation constants showed no major decrease in the Cd-binding affinity in any of the mutants assayed compared with the wild-type.     

Essential functions of synapsins I and II in synaptic vesicle regulation

Synaptic vesicles are coated by synapsins, phosphoproteins that account for 9% of the vesicle protein. To analyse the functions of these proteins, we have studied knockout mice lacking either synapsin I, synapsin II, or both. Mice lacking synapsins are viable and fertile with no gross anatomical abnormalities, but experience seizures with a frequency proportional to the number of mutant alleles. Synapsin-II and double knockouts, but not synapsin-I knockouts, exhibit decreased post-tetanic potentiation and severe synaptic depression upon repetitive stimulation. Intrinsic synaptic-vesicle membrane proteins, but not peripheral membrane proteins or other synaptic proteins, are slightly decreased in individual knockouts and more severely reduced in double knockouts, as is the number of synaptic vesicles. Thus synapsins are not required for neurite outgrowth, synaptogenesis or the basic mechanics of synaptic vesicle traffic, but are essential for accelerating this traffic during repetitive stimulation. The phenotype of the synapsin knockouts could be explained either by deficient recruitment of synaptic vesicles to the active zone, or by impaired maturation of vesicles at the active zone, both of which could lead to a secondary destabilization of synaptic vesicles.     

Inhibition of glycosaminoglycan modification of perlecan domain I by site-directed mutagenesis changes protease sensitivity and laminin-1 binding activity

The effects of medial prefrontal cortex microinjections of 3 nmol/0.5 microl of neurotensin-(1-13), the inactive fragment neurotensin-(1-8), or vehicle on the firing rate of midbrain dopamine neurons were studied in anesthetized rats. Twelve of 19 cells tested with neurotensin-(1-13) showed an average 20-25% increase in firing rate between 10 and 20 min after the injection. This effect was not mimicked by neurotensin-(1-8) (9 cells), nor by a control injection (10 cells) suggesting that it is mediated by high-affinity neurotensin receptors. These results suggest that activation of neurotensin receptors in the medial prefrontal cortex can modulate neural activity of a subpopulation of midbrain dopamine neurons.     

Roles of individual disulfide bonds in the stability and folding of an omega-conotoxin

Although it contains only 25 amino acid residues, omega-conotoxin MVIIA folds into a well-defined three-dimensional structure that is stabilized by 3 disulfide bonds. To assess the contributions of the disulfides to folding and stability, three analogues, each with one pair of disulfide-bonded Cys residues replaced with Ala, were prepared and characterized. The analogues also contained a C-terminal Gly residue that is believed to be present when the peptide folds in vivo and has been shown previously to stabilize the native structure. Circular dichroism spectra and biological assays of the analogues indicated that removing any one of the disulfides greatly destabilized the native conformation. The two disulfides in each analogue were also reduced much more rapidly than in the native form with three disulfides. When the analogues were fully reduced and allowed to form disulfides in the presence of oxidized and reduced glutathione, the native disulfides were not formed in preference to non-native disulfides, further indicating that the forms with two-native disulfides are not significantly stabilized by noncovalent interactions. However, the measured equilibrium constants for disulfide formation indicate that forming any two of the three native disulfides leads to an effective concentration of approximately 25-50 M for the two remaining thiols. The two-disulfide analogues thus appear to represent a stage of folding in which the polypeptide is constrained to a distribution of relatively compact conformations that greatly favor formation of the third disulfide and the final folded structure.     

Characteristics of cation binding to the I domains of LFA-1 and MAC-1. The LFA-1 I domain contains a Ca2+-binding site

The crystal structures of the I domains of integrins MAC-1 (alphaM beta2; CD11b/CD18) and LFA-1 (alphaL beta2; CD11a/CD18) show that a single conserved cation-binding site is present in each protein. Purified recombinant I domains have intrinsic ligand binding activity, and in several systems this interaction has been demonstrated to be cation-dependent. It has been proposed that the I domain cation-binding site represents a general metal ion-dependent adhesion motif utilized for binding protein ligands. Here we show that the purified recombinant I domain of LFA-1 (alphaLI) binds cations, but with significantly different characteristics compared with the I domain of MAC-1 (alphaMI). Both alphaLI and alphaMI bind 54Mn2+ in a conformation-dependent manner, and in general, cations with charge and size characteristics similar to Mn2+ most effectively inhibit 54Mn2+ binding. Surprisingly, however, physiological levels of Ca2+ (1-2 mM) inhibited 54Mn2+ binding to purified alphaLI, but not to alphaMI. Using 45Ca2+ and 54Mn2+ in direct binding studies, the dissociation constants (KD) for the interactions between these cations and alphaLI were estimated to be 5-6 x 10(-5) and 1-2 x 10(-5) M, respectively. Together with the available structural information, the data suggest differential affinities for Mn2+ and Ca2+ binding to the single conserved site within alphaLI. Antagonism of LFA-1, but not MAC-1, -mediated cell adhesion by Ca2+ may be related to the Ca2+ binding activity of the LFA-1 I domain.     

pH- and Ca(2+)-dependent aggregation property of secretory vesicle matrix proteins and the potential role of chromogranins A and B in secretory vesicle biogenesis

Chromogranins A and B (CGA and CGB), the major proteins of the secretory vesicles of the regulated secretory pathway, have been shown to aggregate in a low pH and high calcium environment, the condition found in the trans-Golgi network where secretory vesicles are formed. Moreover, CGA and CGB, as well as several other secretory vesicle matrix proteins, have recently been shown to bind to the vesicle membrane at the intravesicular pH of 5.5 and to be released from it at a near physiological pH of 7.5. The pH- and Ca(2+)-dependent aggregation and interaction of chromogranins, as well as several other matrix proteins, with the vesicle membrane are considered essential in vesicle biogenesis. Therefore, to gain further insight into how vesicle matrix proteins find their way into the secretory vesicles, the pH- and Ca(2+)-dependent aggregation and vesicle membrane binding properties of the vesicle matrix proteins were studied, and it was found that most of the vesicle matrix proteins aggregated in the presence of Ca2+ at the intravesicular pH of 5.5. Furthermore, most of the vesicle matrix proteins bound not only to the vesicle membrane but also to CGA at pH 5.5, with the exception of a few matrix proteins that appeared to bind only to CGA or to vesicle membrane. Purified CGB was also shown to interact with CGA at pH 5.5. The extent and Ca(2+)-sensitivity of the aggregation of vesicle matrix proteins lay between those of purified CGB and CGA, CGB aggregation showing the highest degree of aggregation and being the most Ca2+ sensitive at a given protein concentration. Hence, in view of the abundance of chromogranins in secretory vesicles and their low pH- and high calcium-dependent aggregation property, combined with their ability to interact with both the vesicle matrix proteins and the vesicle membrane, CGA and CGB are proposed to play essential roles in the selective aggregation and sorting of potential vesicle matrix proteins to the immature secretory vesicles of the regulated secretory pathway.     

Site-directed mutagenesis of recombinant human beta 2-glycoprotein I identifies a cluster of lysine residues that are critical for phospholipid binding and anti-cardiolipin antibody activity

beta2-Glycoprotein I (beta2GPI) is a phospholipid-binding serum protein with anticoagulant properties. It plays a vital role in the binding of anti-cardiolipin Abs purified from patients with autoimmune disease when assayed in a cardiolipin (CL) ELISA. Based on a three-dimensional model of beta2GPI, electrostatic calculations, and earlier peptide studies, a highly positively charged amino acid sequence, Lys282-Asn-Lys-Glu-Lys-Lys287, located in the fifth domain of beta2GPI, has been predicted to be the phospholipid binding site. We tested this hypothesis by site-directed mutagenesis of residues in the predicted phospholipid binding site and by assessing the mutants for phospholipid binding and anti-beta2GPI activity. A single amino acid change from Lys286 to Glu significantly decreased the binding of beta2GPI to CL. Double and triple mutants 2k (from Lys286, 287 to Glu286, 287), 2ka (from Lys284, 287 to Glu284, 287), and 3k (from Lys284, 286, 287 to Glu284, 286, 287) possessed no binding of Ab to beta2GPI in a CL ELISA, as well as no inhibitory activity on the binding of iodinated native beta2GPI to CL. These results indicate that the residues Lys284, Lys286, and Lys287 in the fifth domain of beta2GPI are critical for its binding to anionic phospholipids and its subsequent capture for binding of anti-beta2GPI Abs.