Neuroscience Research, 16 (1993) 73-77 73 © 1993 Elsevier Scientific Publishers Ireland, Ltd. All rights reserved 0168-0102/93/$06.00

NSR 00612

U p d a t e ar t ic le

Neurocalcin family: a novel calcium-binding protein abundant in bovine central nervous system

H i r o y o s h i H i d a k a and K a t s u o O k a z a k i

Department of Pharmacology, Nagoya University School of Medicine, Showa-ku, Nagoya 466, Japan

(Received 12 November 1992; accepted 10 December 1992)

Key words: Neurocalcin; Calcium-binding protein; EF-hand; Brain; Central nervous system; Retina; Adrenal gland; Signal transduction

Summary

We purified and sequenced from bovine brain a novel calcium-binding protein. This protein which we named neurocalcin has 3 putative EF hand motifs and a close homology with recoverin which activates guanylate cyclase Ca 2÷ dependently. Neurocalcin has at least 6 isoforms and is expressed in the central nervous system (CNS), retina and adrenal gland. Considering unique distribution of neurocalcin, this protein may an important physiological role which differs from that of visinin or recoverin.

Introduction

Calcium ion (Ca 2+) plays an important role as a second messenger in signal transduction systems in the cell. Ca 2+ receives messages, and sends information to calcium-binding proteins which, in turn, modulate a wide variety of Ca 2 +-dependent events through interac- tion with enzymes and cytoskeletal proteins in the cell. For example, calmodulin exists in almost all cells and modulates many enzymes.

While many calcium-binding proteins have been dis- covered, the functions of these proteins have remained unclear except for calmodulin. In 1977, we synthesized calmodulin antagonists and protein kinase inhibitors, such as W-7 (Hidaka et al., 1978), and these com- pounds have been of great value in the research of calcium-binding proteins and protein kinases. With these conpounds as affinity ligands, we purified and identified several novel calcium-binding proteins, such

Correspondence to." Hiroyoshi Hidaka, Department of Pharmacol- ogy, Nagoya University School of Medicine, Showa-ku, Nagoya 466, Japan.

as calgizzarin (Todoroki et al., 1991) and calvasculin (Watanabe et al., 1992). As such these calcium-binding proteins have tissue- and cell-specific distributions, they may have cell-specific roles differing from those related to calmodulin or S-100 protein (Table 1). Neurocalcin is a family of intracellular calcium-receptive proteins and is abundant in the central nervous system (CNS).

Purification of neurocalcin

Neurocalcin can be purified from bovine brain by an affinity column using the calmodulin antagonist, W-77 ((S)-P-(2-aminoethyloxy)-N-[2-(4-benzyloxy-carbonylpi- perazinyl)-l-(P-methoxybenzyl)ethyl]-N-methylbenzene sulfonamide dihydrochloride), as an affinity ligand (Terasawa et al., 1992). The proteins eluted from W-77 affinity column in a calcium-dependent manner were identified with the 23 and 24 kDa neurocalcin by tricine SDS-PAGE. Neurocalcin was eluted from W-77 affinity column with 10 /zM Ca 2+ after the contami- nated calmodulin was extensively washed out with 1 /xM Ca 2+. Two isoforms of neurocalcin, a- and/3-neu-

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8 - * I ~ / R ~ C I N

B 71 7~

8-NEUROCALCIN N3

7"1

MGKQNSKLRPEVMQDLLESTDFTEHEIQEWYKG~LRDCPSGHLSMEEFKKIYGNFFPYGD ***************************--**************

*A****E**VK**E*N*A*LK ***G*F******L***** ************************************************

************************* **L***I***H******* ************************* **A*******

ASKFAEHVFRTFDANGDGTIDFREFIIALSVTSRGKLEQKLKWAFSMYDLDGNGYISKAE ***********************************************************

*N***N******D*K*TPV* **********************************************************

H** **************XRX* *X* ******~*******XRX*

8 - N E U R O C . A I ~ N3

7"1

MLEIVQAIYE~VSSV--MKMPEDESTPEERTEKIFRQMDTNRDGKLSLEEFZRGAKSDPS *************** *******************************************

****IE**** **SK *************************************************

**X******X ********** ***E ********** ********** L*LE

~-NEUROCALC~ IVRLLQCDP S SAGQF *3 ************S**

7"I ******E*****X** 72 *FIK**P*****X**

Fig . 1. Comparison of amino acid sequence among the neurocalcin family. Amino acids identical to ~-neurocalcin (pCalN) are marked by " * "

8 - N E U R O C A L C I N ; p C a l N , a clone with the full sequence of neurocalcin, N 3 ; a clone with the partial sequence, a , /3, 3,1 and 3,2; isoforms named by partial sequences of the native protein.

rocalcin, can be separated by a C18 reverse phase column on the HPLC system from the 23 kDa protein fraction. The 24 kDa protein fraction can also be divided into two components designated neurocalcin 3,1 and 3,2.

Primary structure of neurocalcin

To characterize the primary structure and clarify the function of neurocalcin, we screened a cDNA library from bovine brain using authentic PCR-amplified cDNA as a probe and we obtained a partial clone (N3) and a complete clone (pCalN), later named 8-neuro- calcin) (Okazaki et al., 1992). The cDNA of pCalN consisted of 1844 nucleotides, including the coding region of 582 nucleotides. The calculated molecular mass of 22,284 corresponding to the open reading

flame of the 193 amino acids is in good accord with that of the native protein, as determined using tricine- SDS-PAGE. The deduced amino acid sequences of N3 and pCalN have a high homology but no identity with the other 4 native isoforms (Fig. 1). Therefore. N3 and pCalN seem to be newly identified members of the neurocalcin family. The deduced amino acid sequence of neurocalcin (pCalN) had 3 putative EF-hand motifs (nos. 73-84, 109-120, and 159-170 amino acid posi- tions).

Biochemical properties of neurocalcin

Although neurocalcin was detected as 23 and 24 kDa bands on tricine-SDS-PAGE, the isometric focus- ing of neurocalcin isoproteins yielded 3 isometric bands at pH 5.3-5.5. Calcium overlay examination verified

T A B L E 1

T I S S U E D I S T R I B U T I O N O F E F - H A N D C A L C I U M - B I N D I N G P R O T E I N S

Brain Muscle

Heart Sketetal Smooth

Lung Platelet

Calmodulin + + + + + +

S - 1 0 0 ( a , / 3 ) + + + + + ?

Troponin C - + + - - - Calcyclin - - - + + +

Calvasculin - - - + - +

Recoverin + (retina) . . . . .9

Neurocalcin + . . . . ?

+ , p r e s e n t ; - , not present; ?, not examined.

the calcium binding to neurocalcin. In an equilibrium dialysis study, the results were not satisfactory because the purified isoprotein was insoluble after HPLC. In our preliminary study, neurocalcin did not affect the activity of CaZ/calmodulin PDE when we added to or replaced calmodulin suggesting that neurocalcin has functions different from calmodulin. Although neuro- calcin has 17 residues of serine and 6 threonine through 193 amino acids, this protein was not phosporylated by either protein kinase A or protein kinase C, under usual assay conditions.

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neurocalcin is thought to be a novel protein. The deduced amino sequence of neurocalcin (pCalN) is unique with a strong homology to recoverin (46.5%) and visinin (51.6%), proteins which are abundant in the retina. The amino acid sequence of the calcium bind- ing loop is homologous with calmodulin but not with parvalbumin. The first and second motifs, which con- sist of 12 amino acids, have a close homology to the second (75%) and third (83.3%) motifs and calmodulin, respectively. Neurocalcin may bind more than 2, maybe 3, moles of Ca 2 ÷ per mole of protein.

Homology search for the neurocaicin family

In a homology search, no identical nucleotide or amino acid sequence was found for neurocalcin, hence

Tissue distribution of neurocalcin

A rabbit polyclonal antiserum to purified neurocal- cin was prepared and the tissue distribution of neuro-

Fig. 2. Immunohistochemical localization of neurocalcin in bovine tissues. A: cerebellum. The molecular layer was moderately stained, whereas the Purkinje cell layer was weakly immunoreactive. P, Purkinje cell layer. Bar = 150/~m. B: retina. Amacrine and granular cells were stained strongly, but the photoreceptor layer was not stain. G, ganglion cell; IN, inner nuclear layer; ON, outer nuclear layer. Bar = 60 p.m. C: adrenal

gland. Zona glomerulosa and adrenal medulla were stained strongly. G, zona glomerulosa; M, adrenal medulla. Bar = 700 p.m.

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calcin was examined by immunoblot analysis (Nakano et al., 1992). Immunoreactive bands were detected in the bovine cerebrum, cerebellum, brain stem, spinal cord and retina. Neurocalcin was not detected in bovine heart, lung, liver, spleen, pancreas, intestine, adipose tissues, and testis and Northern-blot analysis showed similar results. In immunohistochemical studies, pyra- midal cells but not glia cells in the cerebral cortex, were stained. In the cerebellar cortex, the molecular and granular layers stained moderately but the Purk- inje cell layer stained weakly (Fig. 2A). Neurocalcin was also expressed in retinal amacrine ceils and gan- glion cells but not in the photoreceptor layer (Fig. 2B). The presence of neurocalcin in zona glomerulosa and adrenal medula should be noticed (Nakano et al., 1993) (Fig. 2C). However, zona fasciculata and zona reticu- laris were not immunoreactive.

Discussions

We purified and sequenced from bovine brain a novel calcium-binding protein, named neurocalcin. The immunohistochemical study demonstrated that neuro- calcin was abundant in CNS and adrenal gland.

Neurocalcin is an unique calcium-binding protein for the following reasons. Firstly, this protein is a newly identified member of 3 EF hand groups. Calcium-bind- ing protein can be separated broadly into 3 groups: EF hand, annexin and other groups (Heizmann and Hun- ziker, 1991). The EF hand group has characteristic calcium-binding sites called the EF-hand motif. Many calcium-binding proteins with the EF-hand have 2 or 4 domains. Visinin and recoverin are members of 3 EF- hand groups, such a parvalbumin. Neurocalcin is thus a new member of calcium-binding protein with 3 EF-hand structures. Visinin and recoverin lack the EF-hand motif in the C-terminal domain, while neurocalcin ap- pears to degenerate in the N-terminal domain as does parvalbumin. These findings are also of interest from the aspect of the evolution of EF-hand proteins.

Secondly, neurocalcin seems to be expressed mainly in the CNS system, at least in the bovine species. Calmodulin is ubiquitously distributed in various tis- sues and modulates various enzymes, while neurocalcin exists in a tissue- and cell-specific manner. While the function of neurocalcin is yet to be determined, we speculate that it is involved in a cell-specific function in the CNS. We also found that neurocalcin is expressed in the zona glomerulosa and adrenal medulla. As neu- rocalcin has a strong homology with 2 proteins which are abundant in the retina, it may play an important

role in vision systems. Recoverin is a 26 kDa calcium- binding protein in rod and cone photoreceptors of the retina, receptors involved in phototransduction mecha- nisms. Light stimulation induces a depletion of cytoso- lic calcium and recoverin activates photoreceptor guanylate cyclase in such a calcium-free form (Dizhoor et al., 1991). Resynthesized cyclic GMP reopens cation channels which have been closed by the force of light, the result being a recovery of the dark state. Visinin is a 24 kDa calcium-binding protein with 3 EF-hand loops and is present in retinal cone cells (Yamagata et al., 1990). The role of this protein is unknown. Recov- erin-related protein named S-modulin was reported to be a calcium-dependent regulator of cyclic GMP phos- phodiesterase in retinal rod cells (Kawamura and Mu- rakami, 1991). Neurocalcin is highly homologous with these 2 proteins, but neurocalcin is expressed in retinal amacrine cells and ganglion cells but not in the pho- toreceptor layer. It is likely that neurocalcin plays a visinin- or recoverin-like role in other neural tissue or a critical role in phototransduction systems in the retina.

As neurocalcin has several isoforms in the same tissue, it belongs to a family of proteins. Six isoforms have so far been identified in the bovine brain. More- over, CBP-18 reported by Manalan and Klee (1984) and 21k-CaBP by Walsh et al. (1984) are closely related to neurocalcin. P23K reported by Takamatsu et al. (1992) and 21 kDa CaBP by Kuno et al. (1992) were isolated from rat brain and were thought to be related to neurocalcin. It is not clear whether each isoprotein exists and works in a different portion in CNS or whether each isoform plays a different role in the same cell. In either case, neurocalcin probably has an impor- tant role in CNS functions.

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