Calcium-binding proteins: basic concepts and clinical implications.

Calcium ions exert their effects in part via interactions with a wide variety of intracellular calcium-binding proteins. One class of these proteins shares a common calcium-binding motif, the EF-hand. A consensus amino acid sequence for this motif has aided the identification of new members of this family of EF-hand proteins, which now has over 200 members. A few of these proteins are present in all cells, whereas the vast majority are expressed in a tissue-specific fashion. The physiological function of a few of these proteins is known to be achieved via a calcium-dependent interaction with other proteins, thereby regulating their activity. Some members, like parvalbumin, calbindin, and calretinin, proved to be useful neuronal markers for a variety of functional brain systems and their circuitries. Their major role is assumed to be buffering, transport of Ca2+, and regulation of various enzyme systems. Since cellular degeneration is accompanied by impaired Ca2+ homeostasis, a protective role for Ca(2+)-binding proteins in certain neuron populations has been postulated. Another protein family are the annexins, members of which interact with phospholipids and cellular membranes in a calcium-dependent manner. In some cases members of the annexin family were even found to interact with EF-hand proteins. Certain annexins have been suggested to be involved in anti-inflammatory response, inhibition of blood coagulation, membrane trafficking or cytoskeletal organization, but several of these functions have been questioned recently. The elucidation of the interactions and functions of the majority of these proteins remains a challenging task for the coming years.     

Genetically encoded intracellular sensors based on fluorescent proteins

Green fluorescent protein from Aequorea victoria and its many homologs are now widely used in basic and applied research. These genetically encoded fluorescent markers can detect localization of cell proteins and organelles in living cells and also cells and tissues in living organisms. Unique instruments and methods for studies of molecular biology of a cell and high throughput drug screenings are based on fluorescent proteins. This review deals with the most intensively evolving directions in this field, the development of genetically encoded sensors. Changes in their spectral properties are used for monitoring of cell enzyme activities or changes in concentrations of particular molecules.     

Calcium-binding proteins in the vorticellid spasmoneme

The proteins of the contractile spasmoneme from Vorticella convallaria, Carcheslium polypinum, and Zoothamnium geniculatum have been extracted in the detergent, sodium dodecyl sulfate (SDS), as well as urea and guanidine hydrochloride (GuCl). After SDS extraction, the molecular weight distribution of the proteins was examined by means of SDS-polyacrylamide gel electrophoresis. Significant amounts of material corresponding to the contractile proteins actin and tubulin are not present. The contractile organelles in the three species examined contain a group of closely related proteins of molecular weight near 20,000, which constitute a major part (40-60%) of the dry mass. The 20,000 mol wt proteins in Zoothamnium bind calcium with high affinity (pK congruent to 6) and are termed "spasmins." By means of urea polyacrylamide gel electrophorsis, it is demonstrated that in Carchesium and Zoothamnium certain spasmin components bind calcium even in the presence of 6 M urea. The binding of calcium in 6 M urea suggests a functional relationship between the spasmins and the calcium-binding proteins of striated muscle which behave similarly. The calcium binding in urea also indicates that the spasmins within a single spasmoneme have different calcium affinities, and this difference in calcium-binding properties may be an important factor in the physiological function of the organelle.     

Calcium-binding proteins in chemoreceptors of Xenopus laevis.

Calcium-binding proteins were investigated immunohistochemically in chemo-receptors of the olfactory epithelium and taste buds of the clawed frog, Xenopus laevis. Calmodulin-, S-100- and calbindin-immunoreactive material were found in sensory cells of the olfactory epithelium; however, parvalbumin-like material was absent in these cells. Taste buds of the palate showed calmodulin-, S-100- and parvalbumin-immunoreactive material in sensory cells, while calbindin-immunoreactive material in supporting cells. Merkel cells, surrounding the base of the taste buds in a ring-like manner, exhibited calmodulin- and S-100-immunoreactive material.     

Calcium-binding proteins in the parathyroid gland. Detailed studies of parathyroid secretory protein

In order to identify calcium (Ca2+)-binding proteins in the parathyroid gland, we used electrophoretic blots of proteins separated by a two-dimensional nondenaturing/denaturing gel system and incubated them with 45Ca2+. Parathyroid secretory protein (PSP) and proteins with approximate molecular weights of 98,000, 88,000, 58,000, and 30,000 were noted to bind Ca2+ in cytosolic fractions from bovine parathyroid, adrenal, and pituitary glands. However, differences in the binding affinity and capacity of the various proteins were observed. PSP displayed a low affinity and high binding capacity for Ca2+. In the presence of 5 mM MgCl2 and 60 mM KCl, native PSP (immobilized on nitrocellulose filters) bound 7.5 mol of Ca2+/mol of protein monomer with an apparent Kd of 1.1 mM. Immunoblotting identified the association of PSP with parathyroid cell membranes in a Ca2+-dependent manner. This property, together with its heat stability, distinguished PSP from other cytosolic Ca2+-binding proteins which were identified. There was also evidence for a Ca2+-dependent protein-protein interaction (aggregation) of PSP present in a Nonidet P-40 extract of cell membranes. The high Ca2+ binding capacity of PSP and its Ca2+-dependent membrane association may be features that make PSP a potentially important protein in secretory cells.     

Calcium-binding properties of two high affinity calcium-binding proteins from squid optic lobe

We have studied the calcium-binding properties of two high affinity calcium-binding proteins from squid optic lobes: one, squid calmodulin (SCaM), similar to bovine brain calmodulin (BCaM), the other, squid calcium-binding protein (SCaBP), distinct (Head, J.F., Spielberg, S., and Kaminer, B. (1983) Biochem J. 209, 797-802). Equilibrium dialysis measurements on the squid proteins (and BCaM) were made at 100 mM KCl in the presence and absence of 3 mM Mg2+, and at 400 mM KCl in the presence of 3 mM Mg2+, which more closely resembles the conditions in the squid. SCaM, SCaBP, and BCaM each bind a maximum of 4 Ca2+ ions/molecule of protein under the ionic conditions tested. SCaBP has a higher affinity than SCaM or BCaM for Ca2+ at 100 mM KCl in the absence of Mg2+. However, in the presence of Mg2+, half-maximal binding to SCaBP occurs at a similar pCa value to that observed with calmodulin. Increasing the KCl concentration reduces the affinity of all three proteins for Ca2+. UV absorption measurements showed that the binding of 4 Ca2+ ions/molecule is necessary to complete spectral changes in SCaBP, compared to two for the calmodulins. While Ca2+ causes perturbations in aromatic chromophores in SCaM and SCaBP, Mg2+ causes a significant perturbation only in SCaBP. These Mg2+-induced changes differ qualitatively from those induced by Ca2+.     

Soluble calcium-binding proteins: parvalbumins and calmodulin from eel skeletal muscle

1. Eel skeletal muscle contains three parvalbumin isoforms. The overall parvalbumin concentration in the muscle is 0.5 mmol kg-1 wet weight. 2. Calmodulin (0.1 mumol kg-1 wet weight) was purified by extraction with ethylenediamine tetraacetate-containing buffer, fractionation with trichloroacetic acid and separation by ion exchange chromatography on DEAE-cellulose and by molecular sieving on Ultrogel AcA 54. 3. Troponin-C-free calmodulin was obtained by fast protein liquid chromatography on a Mono Q column.     

Calcium-binding proteins from human platelets. A study using crossed immunoelectrophoresis and 45Ca2+

Calcium-binding platelet proteins were examined by crossed immunoelectrophoresis of solubilized platelets against antibodies to whole platelets followed by incubation of the immunoplates with 45Ca2+ and autoradiography. When the immunoplates had been pretreated with EDTA at pH 9.0 in order to remove divalent cations, three immunoprecipitates were markedly labelled with 45Ca2+. These corresponded to the glycoprotein IIb-IIIa complex, glycoprotein Ia and a presently unidentified antigen termed G18. These antigens were membrane-bound and surface-oriented. When an excess of EDTA was introduced in the incubation media the results revealed that the glycoprotein IIb-IIIa complex and antigen G18, but not glycoprotein Ia, contained sites with a stronger affinity for calcium than has EDTA at pH 7.4. Immunoprecipitates of the separate glycoproteins IIb and IIIa both bound calcium in the same manner as the glycoprotein IIb-IIIa complex. As another approach, platelet-rich plasma was incubated with 45Ca2+ prior to crossed immunoelectrophoresis of the solubilized platelets. A single immunoprecipitate was weakly labelled. This did not correspond to any of the immunoprecipitates which were visible after staining with Coomassie blue. The labelling of this antigen was markedly increased when the platelet-rich plasma had been preincubated with EDTA and in this case a weak labelling of the glycoprotein IIb-IIIa precipitate also became apparent. No increased incorporation of calcium occurred in any of these immunoprecipitates when the platelets were aggregated with ADP in the presence of 45Ca2+.     

Calcium-binding proteins in the retina of a calbindin-null mutant mouse

Calcium-binding proteins are abundantly expressed in many neurons of mammalian retinae. Their physiological roles are, however, largely unknown. This is particularly true for calcium-modulating proteins (“calcium buffers”) such as calbindin D28k. Here, we have studied retinae of wildtype (+/+) and calbindin-null mutant (–/–) mice by using immunocytochemical methods. Although calbindin immunoreactivity was completely absent in the calbindin (–/–) retinae, those cells that express the protein in wildtype retinae, such as horizontal cells, were still present and appeared normal. This was verified by immunostaining horizontal cells for various neurofilament proteins. In order to assess whether other calcium-binding proteins are upregulated in the mutant mouse and may thus compensate for the loss of calbindin, mouse retinae were also immunolabeled for parvalbumin, calretinin, and a calmodulin-like protein (CALP). In no instance could a change in the expression pattern of these proteins be detected by immunocytochemical methods. Thus, our results show that calbindin is not required for the maintenance of the light-microscopic structure of the differentiated retina and suggest roles for this protein in retinal function.     

Competitive binding of calmodulin isoforms to calmodulin-binding proteins: implication for the function of calmodulin isoforms in plants.

In plants, multiple calmodulin (CaM) isoforms exist in an organism which vary in their primary structures in as much as 32 residues out of their 148 amino acids. These CaM isoforms show differences in their expression patterns and/or target enzyme activation ability. To further understand the biological significance of CaM isoforms, we examined whether CaM isoforms act on specific regulatory targets. In gel overlay assays on various soybean tissue extracts, surprisingly, two soybean CaM isoforms (SCaM-1 and SCaM-4) did not show significant differences in their target binding protein profiles, although they exhibited minor differences in their relative target binding affinities. In addition, both SCaM isoforms not only effectively bound five known plant CaMBPs, but also showed competitive binding to these proteins. Finally, immunolocalization experiments with the SCaM proteins in sections of various tissues using specific antibodies revealed similar distribution patterns for the SCaM isoforms except for root tissues, which indicates that the SCaM isoforms are concomitantly expressed in most plant tissues. These results suggest that CaM isoforms may compete for binding to CaMBPs in vivo. This competitive nature of CaM isoforms may allow modulation of Ca(2+)/CaM signaling pathways by virtue of relative abundance and differential target activation potency.     

Calcium-binding proteins: differential expression in the rat olfactory cortex after neonatal olfactory bulbectomy

Calbindin, parvalbumin, and calretinin, members of EF-hand calcium-binding proteins, play important roles in buffering intracellular calcium ions. These proteins are localized in distinct populations of cells in the olfactory bulb (the primary sensory relay in the olfactory system) and its major synaptic target, the primary olfactory cortex (POC). In the present study, the postnatal expression of these calcium-binding proteins in layer III of POC was quantitatively examined 30 days after neonatal bulbectomy, a manipulation known to cause cell death and neurotransmitter changes. The numbers of both calbindin and parvalbumin-immunoreactive profiles showed significant increases (68% and 163%, respectively), while calretinin-immunoreactive profiles exhibited a 46% reduction. The data demonstrate that the expression of these calcium-binding proteins is regulated in part by the afferent input from the olfactory bulb. Furthermore, the resultant increase in calbindin and parvalbumin expression may provide neuroprotective support necessitated by possible alterations in intracellular calcium ions and other neurochemical factors that accompany neonatal bulb removal.     

Immunoglobulin superfamily receptors: cis-interactions, intracellular adapters and alternative splicing regulate adhesion.

The immunoglobulin domain is a module found in vertebrates and invertebrates. Its ability to form linear rods when deployed in series, combined with its propensity to bind specifically to other proteins has made it ideal for building cell surface receptors and cell adhesion molecules. These features have resulted in the incorporation of immunoglobulin domains into many hundreds of cell surface molecules. Recently three major advances have been made in understanding immunoglobulin receptors. One is the recognition that their intracellular binding partners are likely to link to multiple cell surface molecules, allowing cross-talk or oligomeric complex formation. A second, but related phenomenon, is their participation in cis-interactions on the extracellular surface that regulate signaling or adhesion. The third is the dramatic ability to form dozens to thousands of different isoforms via alternative splicing. Although antibodies may have been the first example of immunoglobulin-domain-containing proteins using cis-interactions to form receptor like molecules, and the grandest instance of diversity production from limited genetic material, these are clearly old ideas in this superfamily.     

Bacterial expression and characterization of proteins derived from the chicken calmodulin cDNA and a calmodulin processed gene

Both normal chicken calmodulin (CaM) and a CaM-like mutant protein have been expressed in bacteria, isolated and evaluated with respect to several physical and biological properties. The mutant CaM is derived from a CaM-like gene that lacks intervening sequences and probably evolved from a CaM-processed gene (Stein, J. P., Munjaal, R. P., Lagacé, L., Lai, E. C., O'Malley, B. W., and Means, A. R. (1983) Proc. Natl. Acad. Sci. U. S. A. 80, 6485-6489). The mutant CaM protein contains 16 of the 19 amino acids encoded by the CaM-like gene. Normal chicken CaM produced in bacteria is identical to rat CaM by all criteria tested except that it is not trimethylated. The protein product of the CaM-like gene has been termed CaML and exhibits properties which are very similar to CaM despite the presence of 16 amino acid substitutions. CaML binds Ca2+ as evidenced by Ca2+-dependent binding to phenothiazine- and phenyl-Sepharose affinity resins and a Ca2+-dependent electrophoretic mobility shift which is similar to but distinct from CaM. CaML cross-reacts with a monospecific CaM antibody and has an immunodilution curve which is identical to bacterially synthesized CaM. Finally, CaML can maximally activate rat brain phosphodiesterase but with altered kinetic parameters as compared to CaM. These data suggest that the nucleotide substitutions in the putative CaM processed gene are not random but are selected to retain CaM-like functions in the encoded protein. Such a mechanism may exist for other processed genes.     

Calcium-binding proteins of boar spermatozoan plasma membranes: Identification and partial characterization

Calcium-binding proteins (CBPs) of boar spermatozoa and boar seminal plasma were identified by using a ⁴⁵Ca overlay technique to detect these proteins on transblots of PAGE-separated proteins. A single CBP (Mr ～ 300 kDa) was detected in seminal plasma. This protein binds specifically to the plasma membrane overlying the principal segment and is removed from sperm during capacitation. The protein was purified for further charac terization by anion exchange chromatography and gel filtration. In addition, six major proteins (30, 35, 38, 42, 52, and 66 kDa) which do not originate from accessory gland secretions were found to be strongly associated with the plasma membrane. Most of these proteins are not integral to the membrane and appear to develop an association with the plasma membrane during cpididymai maturation. Similarly, calmodulin-binding proteins appear to develop strong associations with the plasma membrane during epididymal transit.     

Prokaryotic calcium-binding protein of the calmodulin superfamily. Calcium binding to a Saccharopolyspora erythraea 20 kDa protein.

The EF-hand calcium-binding protein from Saccharopolyspora erythraea has been shown, using 113Cd NMR, to possess three Cd(2+)-ion binding sites. This indicates that of the four EF-hand motifs in the molecule, one (probably site 2) is unable to bind Cd(2+)-ions. Data from the titration of the protein with Ca2+, in the presence of Quin2, were fitted to a curve calculated on the assumption that the protein contains three high affinity Ca2+ binding sites, two of which (pK1 = 8.0, pK2 = 9.0) are strongly cooperative, and one single site (pK3 = 7.5). Preliminary 1H NMR experiments indicate marked structural changes upon Ca(2+)-binding.