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ISSN 0582-9879                                        ACTA BIOCHIMICA et BIOPHYSICA SINICA 2002, 34(4): 411-417                                     CN 31-1300/Q

Cloning and Characterization of a Novel Human Secretory Protein: Secretogranin III

RONG Yi-Ping1，2, LIU Feng2, ZENG Ling-Chun2, MA Wei-Jun2, WEI Dong-Zhi1*,  HAN Ze-Guang2,1*

( 1Research Institute of Biochemistry, State Key Laboratory of Bioreactor Engineering, East China University of Science

and Technology, Shanghai 200237, China; 2Chinese National Human Genome Center, Shanghai 201203, China )

Abstract     We have cloned a new member of the granin family, termed human secretogranin III (SgIII), that encodes 468 amino acid residues. The human SgIII protein possesses an N-terminal signal peptide, seven dibasic sites, and the repeated DSTK sequences. These structure characteristics are similar to other members of secretogranin family. The human SgIII has homologous proteins in mouse, rat, and Xenopus laevis. Genomic organization shows the gene includes 12 coding exons spanning 39 kb of genomic DNA on the human chromosome 15. Human SgIII is expressed widely as showed in Northern blot and its cDNA hybridizes to 2.2 kb and 1.9 kb bands in many tissues, with two additional 4.5 kb and 3.3 kb bands in brain. Subcelluar localization and immunoblotting indicated SgIII was secreted out of cell through trans-Golgi network (TGN). SgIII may take effect in the biogenesis of secretory granules as a helper protein and be involved in the production or release of peptide hormones in the regulated secretory pathway.

Key words    secretory protein; secretory granule; secretogranin III

The granin family includes chromogranin A (CgA), chromogranin B/secretogranin I(CgB/SgI) and chromogranin C/secretogranin II (CgC/SgII), secretogranin III (SgIII), etc. Huttner et al.[1] suggested that HISL-19 antigen and 7B2 are referred to, in the order of their apparent molecular weight, as secretogranin IV(SgIV) and secretogranin V (SgV), respectively. The granins are very hydrophilic proteins with a high proportion of charged, mostly acidic, amino acid residues and they all have some adjacent basic residues that are potential sites for the proteolytic processing to smaller perhaps biologically active peptides[2,3]. Chromogranin and/or secretogranin family members are often concentrated in the matrix of secretory granules. Recent studies have showed they play an important role in the packaging and sorting of secretory products in the trans-Golgi network (TGN), and in the subsequent formation of secretory granules (reviewed by reference [1, 4-7] ). Secretogranins are widely expressed within secretory granules of endocrine, neuroendocrine and neuronal tissue. Given their widespread distribution, the granins have been used as the most useful markers to investigate the presence of densecore granules in neurons of different areas of the mammalian central nervous system[4].

SgIII is the newly found member of the granin family. It was reported originally in rat, while human SgIII has not been studied[8]. We have found a novel pituitary protein previously in the study of gene expression profiling in human tissues of hypothalamus-pituitary-adrenal axis[9]. The protein has been called human SgIII according to its homology to SgIII of mouse and rat. Its homologue in rat has been identified which is present in the storage vesicles of many neuroendocrine cells, especially in the neurons participating in auditory, olfactory and extrapyramidal motor functions, as well as in neurons related to the hypothalamic-pituitary axis[8]. The study of Xenopus laevis SgIII revealed that it is a sulfated protein undergoing proteolytic processing in the regulated secretory pathway.

In this paper we further investigate the characterization of human SgIII, a novel member of granin family. The tagged human SgIII was predominantly localized in the endoplasmic reticulum and secreted out of the COS-7 cell. As demonstrated in Northern blot, human SgIII is expressed in heart, skeletal muscle, kidney, liver and strongly in brain with specific transcripts of higher molecular weight.

1  Materials and Methods

1.1  Identification, sequencing, and sequence analysis of human secretogranin III

Sequencing was performed using the Applied Biosystems Taq DyeDeoxy Terminator sequencing kit and ABI 377 automated sequencer. Computer analysis of sequences was performed with the Wisconsin Package Version 10.0, Genetics Computer Group (GCG), Madison, Wisc and several shared softwares, such as SignalP v.2.0 (http://www.cbs.dtu.dk/services/SignalP-2.0/)[10,11]. Similarity searches were performed using the BLAST program.

1.2  cDNA cloning and expression plasmid

The full-length SgIII coding sequence was amplified by polymerase chain reaction from the cDNA library of pituitary and hypothalamus (Clontech). PCR primers (forward, XbaI, 5′GC-TCTAGAAGCCGAGCGTGGAAGAAT3′; reverse, KpnI, 5′GGGGTACCCAGGCTGCTATAAATGC-GCTT3′) were used. Amplifications were carried out in a PE9700 thermal cycler with 94 ℃ for 3 min, 35 cycles of 94 ℃ for 30 s, 53 ℃ for 40 s, 72 ℃ for 2 min and a final extension at 72 ℃ for 10 min. For mammalian expression, the fragment was inserted into pcDNA3.1(-)/Myc-HisA vector (Invitrogen) to generate a plasmid encoding SgIII with a Myc-His-tag at the C terminus.

1.3  Expression analysis

Human 12-lane multiple tissue Northern (MTN) blot (Clontech), which contain 1mg poly A+ RNA from one of 12 human tissues per lane, was hybridized according to manufacturer's protocol. The full length coding sequence of the SgIII gene generated by PCR amplification, was labeled using Random Primer DNA Labeling Kit Ver.2 (TaKaRa) as a probe. The blot was washed in 2×SSC/0.5 g·L-1 SDS or 0.1×SSC/1　g·L-1 SDS and exposed to phosphor screen (Molecular Dynamics) for 24 h at room temperature.

1.4  Immunoblotting

The culture medium of transfected COS-7 cells were collected while the cells were washed two times with cold PBS (pH 7.4) and lysed with single detergent lysis buffer. The culture medium and the cell lysates were purified using TALON metal affinity resins (Clontech) according to the manufacturer's manual. Proteins were run on 12% SDS-PAGE and transferred electrophoretically onto PVDF membrane (Amersham Life Science). Membranes were incubated with monoclonal antibody to human c-Myc (0.2 g/L, Clontech) following a horseradish peroxidase-linked secondary antibody and detected using chemiluminescent substrate (ECL, Amersham).

1.5  Immunocytochemistry.

COS-7 cells were cultured in 10% fetal bovine serum/DMEM in 5% CO2. Plates seeded in 1.5×105 were grown overnight, and cells were transfected with 2 mg DNA using lipofectamine (Gibco BRL) according to the manufacturer's protocol. Transfected cells were fixed with 2% parafor-maldehyde (PFA) /Triton-X-100 and immunostained with monoclonal antibodies to human c-Myc (0.2 g/L, Clontech) following the fluorescein-conjugated goat-anti-mouse IgG (0.5　g/L, Gibco BRL), respectively. The staining patterns were viewed with a Laica microscope.

2  Results

2.1  Cloning and characterization of the human secretogranin III gene

Human SgIII (accession no. AF453583) gene is an open reading frame of 1 404 nucleotides in length, encoding a 468-amino-acid protein (Fig.1), which has predicted weight of 53 kD and isoelectric point of 4.8. A search of the GenBank database identified three homologues, Mus musculus SgIII (accession no.NM_009130), Rattus norvegicus (accession no.U02983), and Xenopus laevis SgIII (accession no.X92872). The overall identity between human SgIII with the three homologues are in orderly 87%, 87%, and 56% at the amino acid level. Alignment of these sequences is depicted in Fig.2. SgIII showed high identity to other members of granin family in the region of secretogranin motif (325aa-348aa) (Fig.3)[8,12]. Sequence analysis of SgIII showed it has three potential sites (60, 346 and 350 sites) for N-linked oligosaccharides (Asn-X-Ser/Thr). Moreover, a 19-amino-acid region that highly resembles a signal sequence exists in N-terminal by analysis of SignalP.  The analysis results showed the signal peptide probability is 89.7% and max cleavage site probability is 0.355 between position 26 and 27 residues[10,11].  In line with the presence of tyrosine sulfation site in Xenopus laevis[3], human SgIII also contains a putative tyrosine sulfation site at residue 123. Other domains and O-linked oligosaccharide sites are not been found.

Fig.1  Nucleotide and predicted amino acid sequence of human SgIII

The predicted N-terminal signal sequence is indicated in italics, and its putative cleavage site is indicated with an asterisk. Putative poly(A) addition signals are boxed. Tandem basic residues are indicated by line; the repeated DSTK elements are indicated by dot line. The potential poly(A) addition sites are marked with an arrowhead.

Fig.2  Amino acid sequences comparison of human SgIII with other homologs

Identical residues are in black, and conserved substitutions are in gray.

Fig.3  Comparison with secretogranin motif

The regions of secretogranin motif are compared here: hSgIII (residues 325-348), hCgA (human chromogranin A residues 406-427), hSgI (human secretogranin I residues 650-672), hSgII (human secretogranin II residues 488-512). Gaps have been introduced to optimize the alignments.

By searching the human expressed sequence tags (EST) database and comparison with human genome database, we found three potential cDNA forms resulting from possible different poly (A) addition sites in the last exons in the 3′ untranslated region (UTR). The two short forms, terminated in the positions 1 908 bp and 2 017 bp respectively, were coincident with the two SgIII transcripts 2.2 kb and 1.9 kb in rat. The long form cDNA, 3 371 bp in length, was supported by ESTs most from brain.

Since it has been reported that SgIII is concentrated in brain, especially in pituitary, we obtained the gene from pituitary and hypothalamus cDNA library (Clontech) by PCR, then cloned it into pcDNA3.1A.

2.2  Genomic organization of the human secreto-granin III gene

Comparisons of the human SgIII cDNA sequence with GenBank genome database identified a genomic clone NT010204.6 derived from human chromosome 15. The human SgIII locates in chromosome 15q21.3 and has 12 exons and 11 introns. As demonstrated in Fig.4, the 12 exons from the known genomic SgIII clone are spread over more than 39 kb pairs, with the largest intron at approximate 12 kb between the 10th exon and the 11th exon.

Fig.4  Intron-exon structure of the SgIII gene

The genomic SgIII clone is derived from chromosome 15. (accession no. NT_010204.6). The coding exons of human SgIII are depicted as black proportional bars, with gray portions representing noncoding regions and black portion representing coding region. The last exon contains three possible poly(A) addition sites; they are indicated with different patterns. The putative poly(A) addition signals are indicated with an arrowhead. The slash marks indicate the last exon of the longest form of SgIII, which is approximately 1.6 kb.

2.3  Expression analysis

The tissue expression of human SgIII mRNA was examined by Multiple Tissue Northern blot using the full-length SgIII cDNA as a probe. The SgIII was detected strong hybridization to a 2.2 kb band and weaker hybridization to a 1.9 kb target (Fig.5). But in samples from the brain, it is of interest that four mRNA size variants are present, we detected specific hybridization to 4.5 kb and 3.3 kb bands, while 2.2 kb and 1.9 kb bands were very weak. There are similar levels of mRNA expression in small intestine, placenta, colon and apparently higher levels in brain, heart, skeletal muscle, kidney and liver. It has been reported rat SgIII gene gives rise to 2 stable mRNAs 2.2 kb and 1.9 kb,which differ only in their sites of 3′ polyadenylation[8]. Accordingly, we think the 2.2 kb and 1.9 kb mRNAs of human SgIII were possible due to the difference of poly (A) addition sites. The 3.3 kb transcript in brain agreed with the long cDNA form which was assembled by ESTs mainly from the brain.

Fig.5  Distribution of SgIII mRNA in human tissues

RNA blot containing poly(A+) RNA from multiple human tissues was hybridized with 32P-labled human SgIII cDNA as a probe. Human b-actin was used as a control to determine the relative amount of RNA from each tissue.

2.4  Cellular localization of secretogranin III protein

COS-7 cells were transiently transfected with the pcDNA-SgIII plasmid to examine the cellular localization of the c-Myc-tagged SgIII protein. The localization of expressed protein was visualized using the antibody c-Myc 9E10 and secondary antibody goat anti-mouse IgG (FITC labeled). Although a part of synthesized SgIII was secreted into medium, most of expressed proteins remained intracellular. The tagged SgIII was localized mainly in the perinuclear region as well as a prominent tubular network (Fig. 6), a pattern characteristic which is identical with ER[13,14], and no fusion protein was detected in nucleus.

Fig.6  Immunofluorescence detect of SgIII expressed in COS-7 cells

COS-7 cells, which were transfected with pcDNA-SgIII, were stained with anti-c-Myc antibody and visualized with a fluorescein isothiocyanate-conjugated antibody (B). (A) is a common view of the stained cells. Staining profiles were oberved using a Laica microscope. (C) is an overlap of (A) and (B).

SgIII contains a possible cleavable signal peptide, and no hydrophobic transmembrane segment, which is consistent with transfer of the protein into the lumen of the ER. To verify that the N-terminal stretch of hydrophobic amino acids function as a signal sequence, we tested the expression of SgIII by immunoblotting with human growth hormone as a positive control (Fig. 7). Both in cell lysates and in medium supernatants the tagged SgIII could be detected. Non-specific bands were detected in the lanes of intracelluar pcDNA3.1(-)MycHisA and HuGH-Myc-His. The fusion protein in the extracellular fluid migrated at approximately 63 kD that is greater than the intracellular protein. Previously, the data base search showed SgIII has three potential N-glycolysation sites, so this result indicates SgIII may has been glycosylated during the process of secretion.

Fig.7  Immunoblotting analysis of the tag SgIII expressed in COS-7 cells

The expression of the tagged SgIII in COS-7 cell was examined by immunoblotting with human growth hormone (HuGH-Myc-His) as a positive control and pcDNA3.1(-)MycHisA as a negative control.

3  Discussion

In this study we have characterized human SgIII gene, encoding a 468-amino acid protein with an N-terminal signal sequence. The abundance of acidic residues in the polypeptide leads to an acidic isoelectric point that may contribute to the SgIII taking effect in the low pH environment of trans-Golgi network (TGN). It is known that other established granins, CgA, SgI and SgII also have many acidic residues and low pH can promote the calcium-induced aggregation of human SgII[14,15]. The negative charges of human SgIII may favor its function in the acidic physiological lumenal milieu of secretory granules. Additionally, human SgIII has seven pair dibasic sites that are potential cleavage sites in the posttranslational modifications by endoproteolytic enzymes[2]. Sequence alignment with the SgIII homologues indicates that human SgIII is conserved at DSTK repeated sequences, that is reminiscent of a repeat present in the trans-Golgi network integral membrane proteins TGN38 and TGN41, a finding more consistent with an intracellular function for this protein[3]. Moreover, SgIII contains the obvious secretogranin motif (Fig.4). The biological significance of this motif remains to be investigated. Subcelluar localization and immunoblotting indicates SgIII is secreted into the  cell supernatants through TGN. This result matches the function that granins participate in the packaging and sorting of some neuropeptides and the formation of secretory granules in the TGN[4,17].

Although human SgIII shares many structure features with other homologues and members of granin family, the dibasic sites of human SgIII are not consistent with those of other homologues, such as Xenopus laevis SgIII[3]. Additionally, its tissue distribution is more widely and two specific 4.5 kb and 3.3 kb transcripts only exist in brain. In contrast, rat SgIII is expressed specifically in brain with two variants of 2.2 kb and 1.9 kb[8]. We have found three cDNA forms of human SgIII by EST analysis. Poly(A) tail could appear behind the sites of 1 908, 2 017 and 3 314 bp of this gene respectively, that will generated at least three transcriptional products represented 1.9 kb, 2.2 kb and 3.3 kb hybridization bands in Northern blot. The number of  ESTs represented the 2.2 kb transcriptional product was much more than that of 1.9 kb one. So the 2.2 kb cDNA was predominant and might play primary role in most of human tissues examined while the 1.9 kb transcript might be appurtenant (coincidentally, the ESTs related to the 2.0 kb cDNA form are predominant in most of human tissues). And most of ESTs related to the 3.3 kb form are just from brain. So it can illustrate the result of strong and specific hybridization to 3.3 kb band only in brain. We cannot find other cDNA form correlated to 4.5 kb band. But we still can predict that this 4.5 kb transcript, as the 3.3kb one, is possible due to the different transcriptional termination in the last exon or a special splicing pattern of pre-mRNA. Despite the variation in the 3'UTR, the CDS of human SgIII gene keep constant. Since splicing of pre-messenger RNA is regulated differently in the brain compared with other tissues and it is significant in many cases[18], further work will be required to find the role of brain specific transcripts.

Genetic ablation of the SgIII gene in mice had not obvious effects on viability, fertility or locomotor behavior[19,20]. Many of the cell types that normally express SgIII can survive and function without this protein, perhaps because the normal functions of SgIII can be replaced by the products of other genes. Joost et al. have reported the mRNA levels of Xenopus laevis SgIII in intermediate pituitary increased in parallel with that of proopiomelanocortin when changing the background color of the toad[3,21]. This finding shows SgIII may have a role in the production and release of peptide hormones. Moreover, the endoproteolytic processing of SgIII is a widespread phenomenon in the neuroendocrine system of vertebrates. SgIII may be trigger complex dissociation in its processing and facilitating maturation of the granular contents as a helper protein. Since the function of SgIII is still elusive, further investigation is needed to determine the role of SgIII in the regulated secretory pathway.

Acknowledgements    We acknowledge Dr. Zhang X for her help with our work. We also thank Dr.Xu WQ at Institute of Neuroscience, Chinese Academy of Sciences for photographing.

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Received: January 23, 2002    Accepted: April 3, 2002

This work was supported by the grant from State 863 High Technology R&D Project of China(No.102-08-08-01)

*Corresponding authors: HAN Ze-Guang: Tel, 86-21-50801325; Fax, 86-21-50800402; e-mail, [email protected]; WEI Dong-Zhi: Tel, 86-21-64250068; Fax, 86-21-64250068; e-mail, [email protected]