Biosci. Biotechnol. Biochem., 74 (10), 2050–2055, 2010

Aspartyl , Encoded by an Evolutionarily Conserved Syntenic , Is Colocalized with Its Cluster in Secretory Granules of Pancreatic Islet Cells

y Wendy W. CAI,1;2 Lin WANG,3 and Yuanxiu CHEN1;

1National Center, Howard University College of Medicine, Washington, DC 20059, United States 2International Baccalaureate (IB) Program, Richard Montgomery High School, Rockville, Maryland 20850, United States 3Huazhong Normal University, Wuhan 430079, China

Received May 10, 2010; Accepted July 26, 2010; Online Publication, October 7, 2010 [doi:10.1271/bbb.100349]

Aspartyl aminopeptidase (DAP), encoded by the granule membrane protein in islet cells and a major DNPEP gene, is believed to be a cytosolic protein with autoantigen in Type 1 diabetes.5,6) It is composed of a high enzymatic activity in the neuroendocrine tissues. cytoplasmic region (a.a. 600–979) and a luminal region Bioinformatic analysis revealed that the genomic seg- (a.a. 1–576), which has significant homology at a.a. ment spanning the DNPEP gene is evolutionarily 1–200 with RESP18.7) Ptprn-deficient mice showed conserved from Caenorhabditis elegans to humans. In reduced insulin secretion from isolated islets and glucose the present study, we sought to determine whether the intolerance.8) PTPRN is evolutionarily conserved from expression of DAP is associated with its clustered C. elegans to humans9) and a mutation of the PTPRN when expressed in pancreatic islet cells. Using anti-DAP orthologous gene in C. elegans also impaired the insulin- specific antibody in immunofluorescent stainings, we like pathway.10) found that DAP was specifically expressed in islet alpha Sequence analysis and mapping re- cells but not in exocrine acinar cells. Moreover, using vealed that PTPRN was clustered with RESP18 at electron microscopy, we found that DAP was associated human chromosome 2q35.7) Further investigation con- with a lysosomal-like structure and secretory granules, firmed that RESP18 also encoded a secretory granule suggesting that it plays an important role in post- protein in islet cells and formed a syntenic block with translational processing and the secretion of hormones PTPRN across the mammalian species examined,7) but in islet cells. The identification and characterization of not in invertebrates. DNPEP, another neighbor gene of DNPEP syntenic genes confirm that conserved clustered PTPRN, encodes (DAP, genes can preferentially be expressed in the same EC 3.4.11.21), a mammalian homolog of the M18 signaling pathway. family in yeast, which is believed to be a cytosolic protein with high enzymatic activity in neuroendocrine Key words: aspartyl aminopeptidase; pancreatic islets; tissues. Brain aspartyl aminopeptidase, which converts secretory granules; comparative genomics; angiotensin I to angiotensin 2–10, was identified as a synteny possible target for antihypertensive therapy.11) In this present study, we found that DNPEP was mapped to the Many homologous genes are clustered in genomes same block as PTPRN, and that DAP was co-localized and evolutionarily conserved in syntenic segments with glucagon in secretory granules in pancreatic islet cross-species,1,2) including immunoglobulin genes3) and cells, suggesting that genes from this syntenic block also chemosensory receptor genes.4) As more genomes are function coordinately. sequenced, more syntenic clustered genes will be identified through comparative genomics, which will Materials and Methods dramatically enhance understanding of the significance of functional genomes in the physiology and patho- Bioinformatic analysis. The entire sequence and predicted genes of physiology of life. human DNPEP-containing BAC clone (GenBank acc. no. AC053503) were analyzed by BLAST searching the National Center for Bio- Since Type 1 diabetes is associated with islet cell technology Information databases (http://ncbi.nlm.nih.gov/BLAST) dysfunction, understanding how hormones such as with BLAST algorithms.12) The adjacent genes of DNPEP, including insulin and glucagon are produced and regulated for PTPRN and RESP18, were investigated to determine whether the secretion in the islets of the pancreas is important for the orthologs could be mapped to the vicinity of DNPEP locus in the prevention and treatment of Type 1 diabetes. Thus, genome of other species. Domains and motifs were predicted with bioinformatic analysis and subsequent localization stud- SMART (http://smart.embl-heidelberg.de). C. elegans ies of clustered genes related to islet function may The loci conservation of syntenic genes shared between and humans was determined by database search (NCBI, Ensembl, provide clues for further studies of their roles in glucose Xenbase G-Browse, Ensembl Metazoa, and Wormbase) the genome metabolism. PTPRN (Protein Tyrosine Phosphatase assemblages of 12 species, including seven mammals (human, Homo Receptor type N, gene ID number 5798) is a secretory sapiens; chimpanzee, Pan troglodytes; monkey, Macaca mulatta; cow,

y To whom correspondence should be addressed. Fax: +1-202-806-2265; E-mail: [email protected] Aspartyl Aminopeptidase and Secretory Granules of Islets 2051

Bos taurus; dog, Canis familiaris; rat, Rattus norvegicus; mouse, Mus PTPRN RESP18 DNPEP musculus), one bird (chicken, Gallus gallus), one amphibian (western Human clawed frog, Xenopus tropicalis), one fish (zebra fish, Danio rerio), (Chr.2q35) two insects (fruit fly, Drosophila melanogaster; mosquitoes, Anopheles Chimpanzee (Chr.2b) gambiae), and one worm (nematode, Caenorhabditis elegans). Monkey (Chr.12) Cow (Chr.2) Dog (Chr.37) Antibody and microscopy. Antibody to DAP was the gift of Dr. S. Rat (Chr.9q33) Wilk. Antibody to RESP18 was the gift of Dr. M. Schiller. Antibody to Mouse PTPRN was purchased from Santa Cruz Biotechnology (Santa Cruz, (Chr.1) CA). Anti-insulin, anti-glucagon, and anti-somatostatin mouse mono- clonal antibodies were from Sigma (St. Louis, MO). Paraffin embedded Chicken slides of mouse pancreas tissue were prepared, and immunofluorescent (Chr.7) stains were performed by standard methods. Microscopic analysis was Xenopus done with a Zeiss Axiophot microscope (Columbus, OH). tropicalis missing The Allan Brain Atlas. The Allan Brain Atlas (ABA) (http:// Zebrafish www.brain-map.org) supports gene-specific expression analysis in the (Chr.6) (Chr.9) mouse brain by providing an automated high throughput in situ duplicated hybridization internet platform.13) In situ hybridization data for the C.elegans expression of each gene in adults were extracted from the ABA, a (Chr.III) publicly accessible online digital library widely cited in the literature. All ABA image-based, in situ hybridization data Fig. 1. Syntenic Segment Encompassing PTPRN, RESP18, and DNPEP. are spatially registered to the common anatomic framework of the Each box represents individual genes. Synteny conservation is ARA14) with a standard coordinate system and hierarchical ontology. highlighted by the same color between various species. The synteny The Anatomic Gene Expression Atlas (AGEA) in principle enables structures in all five species (chimpanzee, monkey, cow, dog, and users to view a comprehensive list of genes expressed in any given rat) between human and mouse are similar to that in human and three-dimensional voxel of the mouse brain atlas by using the Gene mouse, and hence are skipped, as indicated by broken arrows. The 15) Finder application. Expression levels in different brain regions orthologous genes in non-mammal vertebrates, including chicken, were downloaded as xml files and processed with Microsoft Excel xenopus, and zebrafish, are also clustered. The orthologous genes in 2007. Sagittal planes of the spinal cord were taken at the cervical the invertebrate C. elegans were intervened by 17 bystander genes spinal level (C1) for further analysis of Dnpep, Resp18, and Ptprn (not displayed). The name of each gene is given on the top row. The expression data. names of species and chromosome numbers (in parentheses) are given on the left. Immunoelectron microscopy. Mouse pancreatic islets were isolated, fixed, and embedded by standard methods.7) Immunogold Electron Microscopy was performed as follows: Briefly, the sections were Results exposed to blocking solution containing 2% BSA, 2% normal goat serum, and 0.1% fish gelatin in PBS containing 0.05% Tween-20 DNPEP was tandemly arranged with PTPRN in a (PBST) for 60 min, and incubated with anti-DAP polyclonal rabbit conserved syntenic region antibody (1/10 dilution) and anti-glucagon monoclonal mouse anti- That PTPRN was clustered with RESP18 across body (1/50 dilution). The primary antibodies were detected with 5-nm mammalian species and matched the sequence at the diameter colloidal gold particles conjugated to goat-anti-rabbit IgG and 10-nm diameter colloidal gold particles conjugated to goat-anti-mouse first 200 amino acids in the luminal region, and was IgG respectively (Ted Pella, Redding, CA). expressed in the same subcellular location in the islet cells of the pancreas, prompted us to hypothesize that Protein fractionation. The synaptosome, which contains vesicles, the conserved region of PTPRN has additional genes was enriched as previously described.16) Briefly, the whole mouse brain sharing properties, including a similar expression pat- was homogenized in buffer (0.32 M sucrose, 20 mM HEPES, pH 7.4, tern. Hence, we analyzed the 163-kb PTPRN-containing with inhibitor cocktail, Sigma) and centrifuged at 1;000 g for BAC clone (RP11–747C8) on human chromosome 10 min to pellet the membrane fragments and nuclei. The supernatant 2q35. We found that orthologs of PTPRN, RESP18, was then centrifuged at 17;000 g for 15 min to obtain pellet-containing synaptosomes contaminated with mitochondria and microsomes. This and DNPEP, clustered in human genome, were also crude synaptosome fraction was further purified using a discontinuous present in the same order on mouse chromosome 1. A sucrose density gradient consisting of a 0.8-M sucrose layer on the top similar syntenic segment (PTPRN-RESP18-DNPEP in and a 1.2-M sucrose layer on the bottom. The pure synaptosomal order) was found in all seven of the mammalian species fraction was obtained from the interface of 0.8-M and 1.2-M sucrose we examined, including Homo sapiens (chromosome after 90 min of centrifugation at 54;000 g. The lysosome- or nuclei- 2q35), Pan troglodytes (Chr. 2b), Macaca mulatta (Chr. containing fraction was then enriched with other appropriate reagents 12), Bos taurus (Chr. 2), Canis lupus familiaris (Chr. (Instructions 89839 and 89841 respectively, Pierce, Rockford, IL). 37), Rattus norvegicus (Chr. 9q33), and Mus musculus (Chr. 1) (Fig. 1). To determine whether the syntenic DAP enzymatic activities. Membrane-bound DAP activity was measured as previously described in buffer of 50 Tris–HCl (pH 7.4), block was evolutionarily conserved in non-mammal 17) 1mM MnCl2, and 0.125 mM aspartyl-beta-NA. Relative fluorescence animals, we searched and analyzed genomes in further was converted into pmoles of product using a standard curve, species, including Danio rerio, Drosophila melanogaster, constructed with increasing concentrations of beta-naphthylamine. and C. elegans. The ortholog of RESP18 was not found The results were recorded as units of aminopeptidase per mg of protein in any of the non-mammal species we examined. The of analyzed tissue. One unit of aminopeptidase activity was the amount ortholog of DNPEP was not found in the insect species of that hydrolyzed 1 pmol of aminoacyl-beta-naphthylamide per min. Protein concentrations were measured by Micro BCA Protein of Drosophila melanogaster or Anopheles gambiae. Assay (Pierce). Data were evaluated by one-way analysis of variance However, ortholog genes of PTPRN and DNPEP were (ANOVA), followed by the DMS test for comparisons among more found in the other four examined species, including than two groups. Gallus gallus (Chr. 7), Xenopus tropicalis (Chr. location 2052 W. W. CAI et al.

PTPRN RESP18 A B

DAP GLUCAGON Merge CDE

Fig. 2. DAP Was Expressed in Islet Cells. PTPRN (A) and RESP18 (B) were expressed in pancreatic islet cells with stronger signal in peripheral alpha cells.7) DAP (C) showed an expression pattern similar to that of glucagon-positive cells (D), and completely overlapped the glucagon signal (E), as determined by double stainings with antibodies to DAP and glucagon separately. All images are presented at 20 magnification. unknown), Danio rerio (Chr. 6), and C. elegans (Chr. area where messenger RNAs that encode DAP, RESP18, III), suggesting that PTPRN-DNPEP is a several- and PTPRN proteins are expressed in adult (56 d) mouse million-year-old syntenic segment. We also found that brain tissues, using the Allen Brain Atlas (ABA) data the cluster was duplicated in Danio rerio (Chr. 9 and sets. The probes used for in situ hybridization were Chr. 6), but the Dnpep gene on Chr. 9 of Danio rerio 781 bp of mouse Dnpep (nt 1058–1819, GenBank acc. was missing. In C. elegans, there are 17 other genes in no. NM 001110831), 591 bp of mouse Resp18 (nt 60– the region (less than 100 kb) between Ptprn (also known 650, GenBank acc. no. NM 009049), and 713 bp of as ida-1) and Dnpep (e.g., F01F1.9). No identical mouse Ptprn (nt 1947–2659, GenBank acc. no. expression patterns were identified in the 17 genes NM 008985). The identities and similarities among according to the Worm Database. Moreover, none of these probes were low (<40–45%), with no significance. these 17 genes was syntenically mapped to the PTPRN- However, the expression patterns of Dnpep, Resp18, and DNPEP-containing regions in other species. Ptprn were very similar, and were present in various regions of brain (Fig. 3). Detailed analysis showed that DAP was expressed in the pancreatic islets higher expression levels of Dnpep, Resp18, and Ptprn Since the 50-terminal flanking region of DNPEP were in the midbrain, pons, medulla, thalamus, main showed considerable similarity to those of PTPRN and olfactory bulb, and hippocampus regions. Lower ex- RESP18 (data not shown), it appeared that the expres- pression levels of these genes were found in the sion of these genes is regulated by common transcription subthalamic nucleus, hippocampal region, cerebral factors for their specific expression in islet cells. As cortex, caudate putamen, and cerebellum, as indicated shown in Fig. 2A and B, PTPRN and RESP18 were both in the Fig. 3. expressed in islet cells, with a stronger signal in the Dnpep, Resp18, and Ptprn were expressed in similar peripheral region. Using DAP-specific rabbit polyclonal patterns in all 20 segments of the spinal cord, from the antibody, we performed immunofluorescent staining of cervical to the lumbar spine. As Fig. 4 illustrates, the mouse pancreas sections. Our results indicated that DAP mRNAs of the three genes were similarly distributed in was also localized to the peripheral cells of the islets the uppermost cervical segment of the spinal cord of the (Fig. 2C). This specific pattern suggest that DAP was adult mice, located mainly in neuron-rich regions of expressed in glucagon-positive alpha cells or somatos- gray matter. tatin-positive delta cells. Further analysis confirmed that the DAP fluorescent signal was completely co-localized DAP was found in secretory granules with glucagon, as detected by anti-glucagon monoclonal To examine subcellular localization of DAP in islet antibody (Fig. 2D and E), but not co-localized with cells further, we conducted immunoelectron microscopy insulin beta cells or somatostatin delta cells (not shown). analysis with anti-DAP antibody. Electron micrographs revealed that DAP was associated with lysosomal-like Dnpep was expressed in neuronal tissues in a pattern structures (Fig. 5A) as well as secretory granules in the similar to Ptprn and Resp18 islet alpha cells (Fig. 5B). Quantitative analysis of DAP To determine whether Dnpep expression in neuronal particles per square micrometer in 20 electron micro- tissues is similar to Ptprn and Resp18, we analyzed the scopic images revealed that there were 84 13 particles Aspartyl Aminopeptidase and Secretory Granules of Islets 2053

Dnpep ctx Dnpep

hip cex

mob cp mb my th sn p 850 microns 694 microns

Resp18 ctx Resp18

hip cex

mob cp th mb sn my hy 1293 microns p

Ptprn ctx

hip 839 microns cex mob Ptprn cp mb th sn my hy p 1340 microns

Fig. 3. Representative Images of in Situ Hybridization in the Adult Mouse Brain (sagittal). The mRNAs of Dnpep, Resp18, and Ptprn were detected in similar patterns in various brain regions, including the midbrain (mb), medulla (my), pons (p), thalamus (th), subthalamic nucleus 839 microns (sn), hippocampal region (hip), main olfactory bulb (mob), cerebral cortex (ctx), caudate putamen (cp), and cerebellum (ctx), as judged Fig. 4. Representative in Situ Expression Images of Dnpep, Resp18, by staining in a sagittal section of mouse brain from the Allen Brain and Ptprn mRNAs in Cervical Spinal Cord of Adult Mice. Atlas. Scale bars indicate microns. All of the three genes were expressed mainly in the gray matter shaped like a butterfly, which contains interneurons and motor around the lysosome and 32 9 in secretory granules neurons. Scale bars indicate microns. compared to 9 3 particles in the cytoplasm (mean SD, p < 0:01). By double immunolabeling with rabbit nuclei-enriched fractions were also prepared for enzy- antibody to DAP (5-nm colloidal gold particles con- matic activity analysis. Our data indicated that the jugated to goat anti-rabbit antibody) and mouse antibody highest activities of DAP (-400 units) were detected in to glucagon (10-nm of colloidal gold conjugated with the synaptosomal and the lysosomal fractions (Fig. 6). goat anti-mouse antibody), we found that DAP was Although our electron microscopic analysis did not co-localized with glucagon in islet alpha cells (Fig. 5C). show the presence of DAP in ER and Golgi or nuclei, lower activities were also found in the fractions of DAP enzymatic activities detected in the mouse brain microsome (-142 units) and nuclei (-57 units) (Fig. 6). Whether DAP aminopeptidase activity in the cells It cannot be excluded, however, that the lower activities was consistent with the subcellular distribution found in were to be attributed to small amounts of heterogenous the electron microscopic analysis, we measured DAP fractions of cytosol proteins that contained the DAP enzymatic activities in fractionated mouse brain tissues. protein. Since DAP was found in secretory granules in the islet cells, we assumed that DAP might be co-localized with Discussion vesicles in the neuronal cells of the brain. Synaptosomes that contain secretory vesicles and mitochondria of In the present study, we found that DNPEP expres- presynaptic terminal and postsynaptic membranes were sion was similar to two other tandemly arranged genes, prepared from brain tissues by classical subcellular PTPRN and RESP18, in the same evolutionally con- fractionation techniques. Lysosome-, microsome-, and served syntenic block. We also found that DAP was 2054 W. W. CAI et al.

A B C

Fig. 5. Subcellular Distribution of DAP in Islet Alpha Cells. Thin sections of mouse pancreatic islet tissue were used in immunoelectron microscopy analysis. Anti-DAP rabbit antibody was detected with goat anti-rabbit IgG antibody conjugated to colloidal gold. (A) DAP particles were abundant around the lysosomal-like structure (10 nm colloidal gold) and were also detected in the luminal region of the secretory granules (B, 5-nm colloidal gold), as indicated by arrows. DAP particles were not detected in the mitochondria or nuclei. (C) DAP (5 nm colloidal gold particles, black arrows) was co-localized with glucagon (detected with 10-nm colloidal gold particles conjugated to goat anti-mouse antibody, white arrows) in DCVs. Scale bar, 100 nm.

500 in the origin of developmental innovations.18,19) DNPEP 400 is an evolutionarily conserved gene found from C. elegans to humans. DNPEP was missing in the insects 300 (Drosophila melanogaster and Anopheles gambiae), 200 although PTPRN was present in both insects. Also, it

/mg protein 100 is not uncommon that all other functionally unrelated bystander genes between PTPTRN and DNPEP,asin Units of DAP activity Units of DAP 0 the case of C. elegans, are not clustered with PTPTRN SynaptosomeLysosome Microsome Nuclei or DNPEP and lost in other species after millions of years of evolutionary divergence and selection.19,20) Fig. 6. Subcellular Distribution of DAP Enzymatic Activities in the Mouse Brain. Considering that common ancestors of C. elegans and Values are expressed as units of aminopeptidase per mg protein, zebrafish in vertebrates diverged 450–500 million from three independent experiments (mean SEM). years,9,21) we hypothesize that conserved genes within the same syntenic block keep similar tissue specific expressed in mouse and human islet alpha cells and was expression patterns in neuroendocrine tissues, including subcellularly associated with secretory granules and islets and neuronal tissues in the brain and the spinal lysosomal-like compartments. DAP enzymatic activity cord, and hence are also be involved in similar bio- assay indicated that the highest activities of DAP was in logical functions. Using DAP-specific antibody in the synaptosomal- and lysosomal-enriched cellular immunostaining and electron microscopy, we found fractions of the brain tissues. These findings confirm that DAP was indeed expressed in the hormone that genes located in the same syntenic block can also secretory granules in islet cells, like its neighbors share similar expression patterns and even related PTPRN and RESP18. In situ hybridization analysis biological functions. showed very similar expression patterns for Dnpep, Previous phylogenetic analysis indicated that PTPRN Resp18, and Ptprn in both the brain and the spinal cord. and RESP18 form a conserved syntenic block in Since genes expressed in the same tissue and cellular mammalian but not in non-mammalian species.7) Both localization can have similar regulatory elements in their RESP18 and PTPRN were expressed in secretory highly conserved noncoding elements (HCNEs),20,22) granules of the same islet cells and were transcription- identification of such genomic regulatory blocks (GRBs) ally upregulated upon high glucose stimulation or with specific expression patterns in pancreatic endocrine hyperglycemia.7) In this study, we determined that cells may provide clues to promote further investigation DNPEP was physically linked with PTPRN in the into the corresponding biological functions of these genomes of both humans and C. elegans, suggesting a genes. strong syntenic conservation of the block over the DAP is believed to be a cytosolic enzyme associated evolutionary spectrum of bilaterian animals. Two with the lysosome in mammals23,24) and the vacuoles PTPRN paralog genes (in chromosome 6 and 9 in yeast,25,26) the mammalian DAP is preferentially respectively) were identified after whole-genome dupli- expressed in neurons and neuroendocrine tissues. High cation in the zebrafish. But one DNPEP paralog in activity of DAP was found in the testis, the kidney,23) chromosome 9 was missing. In fact, a loss of paralogs or and the myelinic and synaptosomal fractions of brain ohnologs in zebrafish was frequently observed. This tissues.27) PTPRN and RESP18 are also associated with might play a role in establishing lineage divergence and secretory granules and granule-enriched fractions in the Aspartyl Aminopeptidase and Secretory Granules of Islets 2055 brain and endocrine cells.6,28) Moreover, DAP was 10) Cai T, Fukushige T, Notkins AL, and Krause M, J. Neurosci., reported to convert angiotensin I to angiotensin 2– 24, 3115–3124 (2004). 10,29) and also to degrade angiotensin II to angiotensin 11) Banegas I, Prieto I, Vives F, Alba F, de Gasparo M, Segarra AB, 23) Hermoso F, Duran R, and Ramirez M, J. Renin Angiotensin. III, suggesting that DAP plays a major role in blood Aldosterone Syst., 7, 129–134 (2006). 11) pressure (see a recent review ). PTPRN was recently 12) Altschul SF, Gish W, Miller W, Myers EW, and Lipman DJ, found in the kidney as well. Knockout of the Ptprn gene J. Mol. Biol., 215, 403–410 (1990). in mice resulted in a marked reduction of renin- 13) Lein ES, Hawrylycz MJ, Ao N, Ayres M, Bensinger A, Bernard angiotensin levels in plasma,30) which suggests that A, Boe AF, Boguski MS, Brockway KS, Byrnes EJ, Chen L, DAP and PTPRN play similar roles in regulating blood Chen L, Chen TM, Chin MC, Chong J, Crook BE, Czaplinska pressure in the kidney. The RESP18 protein was also A, Dang CN, Datta S, Dee NR, Desaki AL, Desta T, Diep E, 31) Dolbeare TA, Donelan MJ, Dong HW, Dougherty JG, Duncan found in the kidney tissue by Western blot. Whether BJ, Ebbert AJ, Eichele G, Estin LK, Faber C, Facer BA, Fields DAP is co-localized with PTPRN and/or RESP18 in the R, Fischer SR, Fliss TP, Frensley C, Gates SN, Glattfelder KJ, same cells of the kidney requires further investigation. Halverson KR, Hart MR, Hohmann JG, Howell MP, Jeung DP, Nevertheless, these findings are remarkable because Johnson RA, Karr PT, Kawal R, Kidney JM, Knapik RH, Kuan they suggest that DAP contributes to the catabolism of CL, Lake JH, Laramee AR, Larsen KD, Lau C, Lemon TA, other acidic amino acid-containing peptides, such as Liang AJ, Liu Y, Luong LT, Michaels J, Morgan JJ, Morgan RJ, angiotensin and perhaps glucagon, in many neuroendo- Mortrud MT, Mosqueda NF, Ng LL, Ng R, Orta GJ, Overly CC, Pak TH, Parry SE, Pathak SD, Pearson OC, Puchalski RB, Riley crine cells. Further studies of DAP’s effects on the ZL, Rockett HR, Rowland SA, Royall JJ, Ruiz MJ, Sarno NR, catabolism of hormones and peptides in the secretory Schaffnit K, Shapovalova NV, Sivisay T, Slaughterbeck CR, granules and vesicles might contribute to understanding Smith SC, Smith KA, Smith BI, Sodt AJ, Stewart NN, Stumpf of this phenomena. KR, Sunkin SM, Sutram M, Tam A, Teemer CD, Thaller C, In conclusion, through genomic localization, tissue Thompson CL, Varnam LR, Visel A, Whitlock RM, Wohnoutka specific expression, subcellular localization, and enzyme PE, Wolkey CK, Wong VY, Wood M, Yaylaoglu MB, Young RC, Youngstrom BL, Yuan XF, Zhang B, Zwingman TA, and activity studies, we found that DAP and PTPRN are Jones AR, Nature, 445, 168–176 (2007). co-localized in the secretory granules of pancreatic islet 14) Dong HW, ‘‘The Allen Reference Atlas: A Digital Color Brain cells, signifying that DAP may play an important role in Atlas of the C57BL/6J Male Mouse,’’ Wiley, Hoboken, pp. 1– the post-translational processing and secretion of hor- 376 (2008). mones. Further studies are required to determine the 15) Ng L, Bernard A, Lau C, Overly CC, Dong HW, Kuan C, functional relationships of DNPEP with the other Pathak S, Sunkin SM, Dang C, Bohland JW, Bokil H, Mitra PP, members of the same syntenic block. Puelles L, Hohmann J, Anderson DJ, Lein ES, Jones AR, and Hawrylycz M, Nat. Neurosci., 12, 356–362 (2009). 16) Bai F and Witzmann FA, Subcell. Biochem., 43, 77–98 (2007). Acknowledgments 17) Cheung HS and Cushman DW, Biochim. Biophys. Acta, 242, 190–193 (1971). This work was partially supported by a research grant 18) Postlethwait JH, J. Exp. Zoolog. B Mol. Dev. Evol., 308, 563– from the National Institute of Diabetes and Digestive 577 (2007). and Kidney Diseases (NIDDK), National Institutes of 19) Catchen JM, Conery JS, and Postlethwait JH, Genome Res., 19, Health (NIH) (5R01DK072128-04). We are grateful to 1497–1505 (2009). 20) Kikuta H, Laplante M, Navratilova P, Komisarczuk AZ, Dr. Guofeng Zhang for electron microscopy analytical Engstrom PG, Fredman D, Akalin A, Caccamo M, Sealy I, assistance at the National Institute of Biomedical Howe K, Ghislain J, Pezeron G, Mourrain P, Ellingsen S, Oates Imaging and Bioengineering (NIBIB), NIH. 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