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Insulin-Like Peptide 6: Characterization of Secretory Status and Posttranslational Modifications

Department of Pediatrics (C.L., J.S., R.K.M.), University of Michigan, Ann Arbor, Michigan 48109; Departments of Cell Biology and Physiology (W.H.W., O.A.W.), Medicine (O.A.W.), and Pediatrics (S.F.W., M.A.S.), University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15213; and Department of Pharmaceutical Sciences (R.B.G.), University of Pittsburgh School of Pharmacy, Pittsburgh, Pennsylvania 15260

Address all correspondence and requests for reprints to: Ram K. Menon, M.D., University of Michigan Medical School, 1205 Medical Professional Building Box 0718, 1500 East Medical Center Drive, Ann Arbor, Michigan 48109-0718. E-mail: rammenon{at}umich.edu.

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Insulin-like peptide 6 (Insl6) is a member of the insulin/relaxin superfamily with unknown biological function(s). In the current report, we establish that meiotic and postmeiotic germ cells of the testis are the principal sites of expression of Insl6. Analysis of stably or transiently transfected cells revealed that Insl6 is a secreted protein localized to the endoplasmic reticulum and Golgi. Secretion could be detected in both CHO and GC2 germ cells and was sensitive to brefeldin A treatment. In cell lysates, the predominant Insl6 band was approximately 28 kDa in size. In contrast, the predominant Insl6 species in the supernatant was 8 kDa in size, suggesting posttranslational processing of the precursor protein. Ectopically expressed Insl6 is processed and secreted in furin-deficient LoVo cells and in CHO cells treated with a furin inhibitor, although the size profile of the secreted protein is altered suggesting that Insl6 is a substrate for furin action. Furthermore, mutation of a putative furin cleavage site in the Insl6 peptide resulted in aberrant processing of the Insl6 peptide. Additional investigations of the structure of Insl6 protein provided evidence for posttranslational modifications of Insl6, including the presence of disulfide bonds, glycosylation, and ubiquitination. On the basis of the demonstrated secretory status of Insl6, we speculate that the physical proximity of the germ cell to the Sertoli cell renders the Sertoli cell a likely candidate for Insl6 action.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
THE INSULIN/IGF/relaxin family is an ancient family of functionally diverse proteins. Insulin or insulin-like proteins have been described in unicellular eukaryotes, primitive species such as insects, tunicates, annelids, and molluscans (1, 2, 3, 4, 5, 6). Despite functional divergence within the family, all proteins of the insulin family exhibit a high degree of structural conservation. The primary peptide sequence of each member of the family is characterized by three domains comprised of an amino-terminal B peptide (or chain) joined to a carboxyl A peptide by an intervening C peptide (B-C-A) (7). Between the different hormones of the family and between species for a specific hormone, the B and A chain peptides are relatively invariant and exhibit a pattern of distinct and highly conserved cysteine motifs. These cysteine motifs characterize the family; specifically, the motif present in the A peptide has been termed the insulin signature. Many members of the insulin family of hormones are synthesized as preprohormones with the primary peptide undergoing posttranslational modification to generate a disulfide bond-linked heterodimer of the B and A peptides that functions as the active hormone.

At the beginning of the past decade, the insulin family was comprised of four members in mammals: insulin, IGF-I and IGF-II, and relaxin. Over the past decade additional members of the family termed insulin-like (INSL) 3 (8, 9), INSL4 (10, 11), INSL5 (12, 13), INSL6 (13, 14, 15), and INSL7 (16) have been identified. The precise biological role(s) of many of these new proteins, including Insl6 remains to be determined. Although previous studies had established that the testis is the site of maximum expression of Insl6, there is discordance among published studies as to the cell-type in the testis in which Insl6 is expressed. Hsu (13) localized expression to Leydig cells, whereas Lok et al. (15) concluded that Insl6 is expressed in pachytene spermatocytes and round spermatids but not in Leydig cells. At present, information regarding the secretory status, posttranslational modifications, and processing of Insl6 protein is lacking. The aims of this study were to determine the identity of the cell type-expressing Insl6 in the testis, to investigate the subcellular localization and secretory status of Insl6, and to identify and characterize potential posttranslational modifications and processing of this protein.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Cell culture
The culture media used for tissue culture experiments were obtained from Invitrogen (Carlsbad, CA) unless otherwise stated. Flp-In CHO cells (Invitrogen) and mInsl6-CHO cells were grown at 37 C in F12 medium supplemented with 10% fetal bovine serum (FBS). Germ cell (GC2), CRE8 cells, HEK293, and LoVo cells were grown in DMEM (4.5 g/liter glucose) with 10% FBS.

Generation and characterization of anti-Insl6 antibody
Employing the services of a commercial vendor (BioSource International, Camarillo, CA), antimouse Insl6 sera were raised in the rabbit against keyhole hemocyanin-conjugated peptides termed EE and YV representing amino acid (aa) residues 56–71 and 134–150, respectively; both peptides overlap with the putative C peptide (14). The antisera were affinity purified against the homologous peptide using standard techniques. The specificity of the antibodies was verified by demonstrating that the homologous peptide (200 M excess) was able to block the detection of the Insl6 protein by Western blot analysis, whereas insulin failed to exhibit such competition (data not shown). In certain experiments, an anti-A chain mInsl6 antibody from Phoenix Pharmaceuticals (Belmont, CA) was used.

Animals
Eight-week-old male mice (C57BL) (Hilltop Laboratory Animals, Inc., Scottdale, PA) and testicular tissues from WBB6F1/J-Kit^w/Kit^W-v or WB/ReJ Kit^w/+ mice (The Jackson Laboratory, Bar Harbor, ME) were purchased. Female Sprague Dawley rats carrying pups at 19 d gestation were exposed to a single dose of whole-body x-ray irradiation equivalent to a dose of 200 rads using a ¹³⁷Cs source. Age-matched, nonirradiated females were employed as controls. The pups were born spontaneously on d 22 and killed 9 wk after birth. All experiments were carried in accordance with protocols approved by the Institutional Animal Care and Use Committees at the Children’s Hospital of Pittsburgh, the University of Pittsburgh, or the University of Michigan.

Tissue collection
Euthanized animals were perfused with ice-cold saline followed by 4% paraformaldehyde in 50 mM sodium acetate (pH 6.5) and then with 4% paraformaldehyde in 50 mM Tris-HCl (pH 9.0). Testes were removed and postfixed in 4% paraformaldehyde in phosphate buffer (pH 7.2) at 4 C overnight. Testes were sequentially immersed at 4 C for 24-h periods in phosphate buffer (for paraffin embedding) or in 15% sucrose in phosphate buffer followed by 30% sucrose in phosphate buffer (for collecting frozen sections). Paraffin sections (10 µm), and frozen sections (20 µm) were cut and mounted onto Super Frost Plus glass slides (Fisher Scientific Co., Pittsburgh, PA). Paraffin sections were stored at room temperature and frozen sections were stored at –20 C before staining.

Staining
Paraffin sections were first deparaffinized with xylene and rehydrated with 50 mM PBS. Frozen sections were thawed in a desiccator at room temperature and then hydrated with 50 mM PBS. All sections were treated with 0.3% H₂O₂ in PBS for 15 min to inactivate endogenous peroxidase activity. Tissues were then rinsed with PBS, and primary antibody was applied at a dilution of 1:5000 or 1:10,000 in PBS containing 0.05% Triton X-100 and 5% normal goat serum. Control sections were either not exposed to the primary antibody or were treated with the primary antibody plus blocking peptide (10 µg/ml). Sections were incubated for 3 d at 4 C. Sections were then rinsed with PBS and treated with biotinylated goat-antirabbit IgG (3.0 µg/ml, Vector Laboratories, Burlingame, CA) for 3 h at ambient temperature. Sections were then rinsed with PBS. Antibody binding was visualized using the avidin-biotin-horseradish peroxidase (HRP) Elite system (Vector Laboratories). The avidin-biotin-HRP reagent was used at a concentration of 5 µl/ml each of reagent A and B in 50 mM PBS and incubated for 3 h at ambient temperature. Sections were reacted in a solution containing 3–3' diaminobenzidine (0.5 mg/ml, Sigma-Aldrich, St. Louis, MO), 0.01% H₂O₂, and 0.03% NiCl in 50 mM Tris-HCl (pH 7.6) for 10 min. After antibody staining, sections were rinsed with PBS, some were counterstained with Mayers hematoxylin solution (Sigma-Aldrich), dehydrated with ethanol, cleared with xylenes, and cover slipped with DePeX (Gurr, BDH Laboratory Supplies, Poole, UK). For the tissues that were stained with Cy-3, the biotinylated secondary and avidin-biotin-HRP steps were omitted. Sections were incubated with Cy-3-labeled goat-antirabbit IgG (7.5 mg/ml; The Jackson Laboratory) in 50 mM PBS + 0.05% Triton for 1 h at room temperature. The sections were rinsed with 50 mM PBS, counterstained with Sytox Green (1:5000) for 5 min, and rinsed again with PBS. The sections were then rinsed briefly with water and overlaid with coverslips using Gel/Mount (Biomedia Corp., Foster City, CA) aqueous mounting media.

Plasmid construction and generation of Insl6-expressing stable cell line
The mInsl6-TOPO II and hInsl6-TOPO II constructs were generated by cloning murine or human Insl6 cDNA, respectively, into the TOPOII vector (Invitrogen). The plasmids encoding mInsl6 or hInsl6 fused at the C terminus to the myc-His tag were generated by subcloning Insl6 cDNA into the eukaryotic expression vector, pcDNA3.1(+) myc-his (Invitrogen). CHO cells stably expressing mInsl6 (mInsl6-CHO) were generated using the Flp-In system (Invitrogen) as per the manufacturer’s protocol. Briefly, Insl6 tagged at the C terminus with the myc-his epitope was cloned into the Flp-In expression vector, pcDNA5/FRT. Flp-In CHO host cells were cotransfected with Insl6-myc-his pcDNA5/FRT and the Flp recombinase expression vector pOG44. Isogenic expression cell lines were isolated using hygromycin selection.

Recombinant adenovirus expressing murine Insl6
The myc-His epitope-tagged mouse Insl6 gene was subcloned into at the SmaI site of the pAdlox plasmid (Somatix Therapy Co., Alameda, CA) (17). Three micrograms each of the recombinant pAdlox plasmid and the 5 helper virus was transfected into Cre8 cells using Lipofectomine (Invitrogen). The culture medium was replaced every 2 d and the cells along with the media harvested in 7–8 d when the majority of the cells had rounded or detached. Cre8 cells were reinfected twice with the cell lysate to purify and amplify the recombinant virus (Ad-mInsl6). Virus titer was determined by plaque assay in HEK293 cells.

Production of recombinant mInsl6 with mutation in putative furin cleavage site
The putative furin cleavage motif (RKRR) in mInsl6 peptide located between aa 162 and 165 was mutated (to AAAA) using the QuickChange Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA) according to the manufacturer’s instructions.

Immunofluorescence staining
mInsl6-CHO stable cells or Ad-mInsl6-infected GC2 cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100. Double staining with rabbit or mouse anti-myc (1:100 dilution) and rabbit anti-Grp78 (1:100) or mouse GM130 (1:100) antibody was visualized by fluorescein isothiocyanate (FITC)-labeled antimouse IgG and Rhodamine-labeled antirabbit IgG. Cells were observed using a confocal microscope (Olympus FluoView 500, Olympus, Tokyo, Japan) or a Nikon TE200 Eclipse fluorescence microscope (Nikon, Tokyo, Japan). In certain experiments, the cells were exposed to nocodazole (20 µM for 60 min on ice) before fixation and immunofluorescence staining.

Transient transfection
CHO cells (0.4 x 10⁶) were plated on 35-mm plates 24 h before transfection. Plasmid DNA (3 µg) was transfected per plate using the Lipofectamine method (Invitrogen). After 6 h of incubation, the DNA-Lipofectamine mixture was removed and then supplemented with medium for 48 h before harvest for Western blot analysis.

Infection of GC2 cells with Ad-mInsl6
GC2 cells were infected with Ad-mInsl6 at 30 plaque-forming units/cell. In indicated experiments, infected cells or supernatant from the infected cells were harvested after 48 h of exposure of the cells to either brefeldin A (BFA) or vehicle.

Deglycosylation assay
Protein extracts [lysed in radioimmunoprecipitation assay buffer containing 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 2 mM EGTA and 0.1% Triton X-100] from CHO cells transiently transfected with Insl6 expressing plasmid or GC2 cells infected with Ad-mInsl6 were treated with peptide N-glycosidase F (PNGase F) or endoglycosidase (Endo) H (New England Biolabs, Beverly, MA) according to the manufacturer’s protocol. For PNGase F treatment, a 10-µl aliquot of protein was mixed with 1 µl 10x denaturation buffer and boiled for 10 min. The denatured protein mixture was mixed with 1 µl 10x reaction buffer, 1 µl 10% NP 40, and 1 µl PNGase F. The reaction was carried out at 37 C for 1–4 h. For Endo H treatment, 30-µg aliquots of protein extracts were denatured with 1x glycoprotein denaturing buffer at 100 C for 10 min and then treated with 500 U Endo H in the presence of 1x reaction buffer [50 mM sodium citrate (pH 5.5)] for 1 h at 37 C. The reactions were stopped by boiling in sodium dodecyl sulfate sample buffer at 100 C for 5 min and analyzed by SDS-PAGE.

Western blot analysis
CHO or LoVo cells transiently transfected with Insl6 plasmid construct or CHO cells stably expressing Insl6 were harvested at indicated time points. GC2 cells infected with Ad-mInsl6 were harvested after 48 h of infection. BFA (Sigma-Aldrich) treatment was performed by exposing cells to BFA (1–50 µg/ml) for the indicated time periods. Cells were washed twice with cold PBS, lysed in radioimmunoprecipitation assay buffer [50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 2 mM EGTA, and 0.1% Triton X-100] supplemented with a protease inhibitor cocktail (Roche Diagnostics, Indianapolis, IN), supernatant precipitated with 15% trichloroacetic acid, and the protein pellet resuspended in 1% sodium dodecyl sulfate. Equal amounts protein were size-fractionated via SDS-PAGE and subjected to Western blot analysis as previously described (18). The primary antibodies used in these analyses were anti-myc (9E10, Santa Cruz Biochemicals, Santa Cruz, CA) routinely used at a dilution of 1:500. All results are representative for three or four independent experiments.

Lactate dehydrogenase activity assay
Cytotoxicity was determined using a cytotoxicity detection kit (Roche Diagnostics). Briefly, 100 µl supernatant aliquot was mixed with 100 µl reaction mixture and incubated for 20 min at room temperature. Low and high controls for the assay were established by assaying the cell culture medium and the supernatant of Triton X-100-treated cells, respectively. The cytotoxicity index (CI) was determined by the equation: CI = (sample – low control)/(high control – low control) x 100.

In vitro transcription and translation
Transcription and translation coupled reticulocyte lysate and wheat germ extract systems were purchased from Promega (Madison, WI). The reactions were initiated by mixing 3 µg Insl6-TOPO II plasmid DNA with 50 µl reticulocyte lysate or wheat germ extract reaction mixtures in the presence of [³⁵S] cysteine (MP Biomedicals, Irvine, CA) and T7 polymerase. After incubation at 30 C for 90 min, the products were size-fractionated via electrophoresis through a 14% SDS-PAGE gel. In some reactions, His-ubiquitin (Sigma-Aldrich) was added at the initiation of the reaction (19). Where indicated, the intensity of the specific bands was quantitated by densitometry (Bio-Rad, Hercules, CA).

RNA extraction
Total RNA was extracted from testes either via a guanidine isothiocyanate-based technique followed by cesium chloride gradient purification or by using TRI-Reagent (Molecular Research Center, Cincinnati, OH) (20).

Real-time RT-PCR assay
Real-time quantitative RT-PCR using the ABI Prism 7700 Sequence Detection System (PE Biosystems, Foster City, CA) was carried out using established protocols (21). The primers (synthesized by Invitrogen) and TaqMan probes (synthesized by PE Biosystems) for the quantitation of the Insl6 and LH receptor transcripts were designed using the primer design software Primer Express (PE Biosystems) (Table 1). The primers and TaqMan probe for 18S rRNA were purchased from a commercial vendor (PE Biosystems). The 18S probe was labeled with the reporter fluorescent dye VIC and the Insl6 and LH receptor probes with the reporter dye 5-carboxyfluorescein (FAM). The relative efficiencies of the Insl6 and LH receptor primers/probe sets and the 18S primer/probe pair were tested by subjecting serial dilutions of a single RNA sample from each of the tissues analyzed to real-time RT-PCR analysis. The plot of log input vs. Threshold cycle (C_T) was less than 0.1, which satisfies the previously established criterion for equivalence of efficiency of amplification (22). After confirming that the efficiency of amplification of the gene of interest (e.g. Insl6) and 18S transcripts were approximately equal, the amount of the transcripts for the specific gene relative to the 18S transcript was determined by using the comparative C_T (separate tube) method (22). Briefly, 5-ng aliquots of total RNA were analyzed using the One-Tube RT-PCR protocol (PE Biosystems). After RT at 48 C for 30 min, the samples were subjected to PCR analysis using the following cycling parameters: 95 C for 10 min and 95 C for 15 sec 60 C for 1 min for 40 cycles. Each sample was analyzed in triplicate in individual assays performed on two or more occasions.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

Immunocytochemistry.
For purposes of immunostaining with anti-Insl6 antibody, we evaluated both paraffin and frozen testis sections. The staining observed under both conditions was similar with the exception that staining of the frozen sections tended to be denser. Using an antipeptide antibody (EE) directed against an epitope of the Insl6 encompassing contiguous portions of the B and C chains of the Insl6 molecule, we observed staining of the germ cells of the testis. The germ cells stained with the Insl6 antibody were midpachytene spermatocytes, secondary spermatocytes, and round spermatids (Fig. 1A). Preincubation of the antibody with the homologous peptide abrogated the staining indicating that the observed staining was specific (Fig. 1B). Counterstaining with a nucleus-specific stain (Sytox green, Invitrogen) indicated that in germ cells, Insl6 was excluded from the nucleus and was localized to the cytoplasmic region (Fig. 1C). No specific staining of either Leydig or Sertoli cells was detected. Similar results (data not shown) were observed with an antipeptide antibody (YV) directed against a second epitope of the murine Insl6 protein.

View larger version (79K): [in this window] [in a new window]   FIG. 1. Immunohistochemical detection of Insl6 expression in germ cells of the testis. Insl6 expression in testicular section from mice (8 wk old) using anti-Insl6 antibody (EE) without (A and C) and with (B and D) presaturation with antigen peptide (x200). The binding of the primary antibody was either visualized by light microscopy using the avidin-biotin HRP methodology (A and B) or via confocal microscopy of sections labeled with Cy-3-conjugated secondary antibody (C and D). The slides were also counterstained with either hematoxylin (A and B) or Sytox green (C and D). The specific stainings of leptotene spermatocytes (L), pachytene spermatocytes (P), spermatids (S), and elongated spermatids (ES) are indicated by solid arrows. The results are representative of two or more independent experiments. Scale bar, 25 µm in A and B; scale bar, 10 µm in C and D.

View larger version (21K): [in this window] [in a new window]   FIG. 2. Effect of germ cell aplasia on Insl6 mRNA and protein expression. A and B, Steady-state abundance of Insl6 mRNA was measured by real-time quantitative RT-PCR assay in testis of rats (A) or mice (B) (n = 3–7). The abundance of LH receptor mRNA was assayed as a marker of germ cells. A, Rats exposed to radiation during fetal life compared with nonexposed control rats. B, Germ cell aplasia mice WBB6F1/J-KitW/KitW-v compared with the control mice, WB/ReJ Kitw/+. The values are represented relative to level of expression in the vehicle-treated group and are depicted as mean and range. The range is determined by evaluating the expression 2–CT with CT + s and CT – s, where s is the SD of the CT value (22 ). *, P < 0.01 compared with the control group by ANOVA.

View larger version (21K): [in this window] [in a new window]   FIG. 3. Insl6 expression: ontogeny and tissue distribution. A, Ontogeny of Insl6 expression in mouse testis. Expression of Insl6 mRNA was measured by real-time quantitative RT-PCR assay in testis of mice (n = 3–5) at indicated ages. The values (mean ± SE) are represented as relative to level of Insl6 mRNA at 90 d designated as 100%. e, embryonic; p, postnatal. B, Tissue-specific expression of Insl6. Insl6 mRNA levels were measured by real-time quantitative RT-PCR assay in the indicated tissues of adult (6–8 wk old; n = 3–5) male (small bowel, kidney, spleen, and thymus) or female (uterus and breast). The levels are depicted as relative to Insl6 abundance in testis designated as 100%. The results are depicted as mean and range. The range is determined by evaluating the expression, 2–CT with CT + s and CT – s, where s is the SD of the CT value (22 ).

Intracellular localization of Insl6.
The subcellular localization of Insl6 was investigated by colocalization studies of both CHO cells stably expressing either mInsl6 or hInsl6 and GC2 cells expressing adenoviral-mediated mInsl6. Staining with antibodies directed against Bip (Fig. 4A) and GM130 (Fig. 4B) proteins as marker for the endoplasmic reticulum (ER) and the Golgi apparatus, respectively, revealed that in both CHO and GC2 cells, Insl6 protein colocalized to the perinuclear ER region and to the Golgi apparatus. Furthermore, exposure of the cell to nocodazole, a reagent that disrupts microtubules and causes fragmentation of the Golgi apparatus, revealed that there was parallel redistribution of GM130 and Insl6, indicative of localization of Insl6 to the Golgi apparatus (Fig. 4B).

View larger version (30K): [in this window] [in a new window]   FIG. 4. Subcellular localization of Insl6 by coimmunofluorescence. A, CHO cells stably expressing either c-myc-tagged mInsl6 (i–iii) or hInsl6 (iv–vi) were fixed, permeabilized, and incubated with primary antibodies, mouse anti-myc, or rabbit anti-Bip. The specific binding of the primary antibodies was detected by using FITC-labeled antimouse IgG or rhodamine-labeled antirabbit IgG, respectively. Immunofluorescence staining for Insl6 (i and iv) and the ER marker Bip 78 (ii and v) depict identical patterns as illustrated in the merged panels (iii and vi). The results are representative of three or more independent experiments. B, GC2 cells exhibiting adenovirus-mediated mInsl6 expression (i–iii) or CHO cells stably expressing c-myc-tagged mInsl6 (iv–ix) were fixed, permeabilized, and incubated with primary antibodies, rabbit anti-myc, or mouse anti-GM130. The specific binding of the primary antibodies was detected by using FITC-labeled antimouse IgG or rhodamine-labeled antirabbit IgG, respectively. Immunofluorescence staining for Insl6 (i and iv) and the Golgi marker GM 130 (ii and v) depict overlapping patterns as illustrated in the merged panels (iii and vi). To further demonstrate the Golgi localization of Insl6, mInsl6-CHO cells were exposed to nocodazole (vii–ix) before fixation and immunofluorescence staining for Insl6 (vii) and the Golgi marker GM130 (viii) as described above. The disruption of Golgi by nocodazole and consequent parallel redistribution of GM 130 and Insl6 are illustrated in the merged panel (ix). The results are representative of two independent experiments.

Insl6 is a secreted protein.
Based on the knowledge that canonical members of the insulin family are secreted proteins, the possibility of Insl6 being a secreted protein was tested by analyzing the supernatant of CHO cells stably transfected with mInsl6 (mInsl6-CHO) and GC2 cells infected with adenovirus expressing mInsl6 (Ad-mInsl6). As shown in Fig. 5, mouse Insl6 protein could be detected in the supernatant of both cell types, supporting the hypothesis that Insl6 is a secreted protein. In contrast to the cell lysate where the predominant species was 28 kDa in size (top), in the cell culture supernatant, a substantial fraction of the secreted Insl6 was 8 kDa in size (bottom). A similar size profile of Insl6 protein was also observed in CHO cells transiently transfected with mInsl6 (data not shown). These results suggest that the 8-kDa form represents a processed form of the precursor 28-kDa species of mInsl6.

View larger version (26K): [in this window] [in a new window]   FIG. 5. Insl6 is a secreted protein. Naïve CHO cells (lane 1), CHO cells stably expressing c-myc-tagged mInsl6 (lane 2), Ad-mInsl6-infected GC2 (lane 3), or naïve GC2 cells (lane 4) were maintained overnight in DMEM supplemented with 2% FBS. The cells (top) and culture medium (bottom) were harvested separately and processed for analysis. Approximately 2% of whole-cell lysate (top) and 20% of the concentrated culture medium (bottom) (from 35-mm culture plate) was subjected to SDS-PAGE size fractionation under reducing conditions and Western blot analysis with anti c-myc antibody for the presence of the Insl6 protein. The predicted compositions of the various Insl6 bands and the positions of the molecular mass marker are indicated.

View larger version (39K): [in this window] [in a new window]   FIG. 6. Secretion of Insl6 is sensitive to BFA treatment. A, CHO cells stably expressing c-myc-tagged mInsl6 were treated with BFA at the indicated concentration for 6 h, and the culture medium (top, lanes 2–7) and cells (top, lane 1; bottom, lanes 1–5) were harvested separately and processed for analysis. Approximately 20% of the culture medium from culture plate (35 mm) was subjected to SDS-PAGE size fractionation under reducing conditions and Western blot analysis with anti c-myc antibody for the presence of the Insl6 protein. The predicted compositions of the various Insl6 bands and the positions of the molecular mass marker are indicated. B, Insl6-adenovirus (Ad-mInsl6)-infected GC2 cells were treated with BFA at the indicated concentration overnight, and the culture medium was harvested and processed for analysis. Approximately 20% of the concentrated culture medium (from 35-mm culture plate) was subjected to SDS-PAGE size fractionation under reducing conditions and Western blot analysis with anti c-myc antibody for the presence of the Insl6 protein. The predicted compositions of the various Insl6 bands and the positions of the molecular mass marker are indicated.

View larger version (27K): [in this window] [in a new window]   FIG. 7. Insl6 is a target for processing by furin. Top, Processing of Insl6 protein in CHO and furin-deficient LoVo cells. Supernatants of CHO cells stably expressing c-myc-tagged mInsl6 (lane 1), CHO cells transiently transfected with furin cleavage site mutant mouse c-myc-tagged Insl6 (lane 2), LoVo cells transiently transfected with wild-type mouse c-myc-tagged Insl6 (lanes 3 and 4), or LoVo cells transiently transfected with furin cleavage site mutant mouse c-myc-tagged Insl6 (lanes 5 and 6) in the absence (lanes 3 and 5) or presence (lanes 4 and 6) of ectopic expression of human furin were size-fractionated by SDS-PAGE under reducing conditions and analyzed by Western blot analysis with anti c-myc antibody for the presence of the Insl6 protein as described in Materials and Methods. Approximately 20% of the culture medium from each transfection was subjected to this analysis. The predicted compositions of the various Insl6 bands and the positions of the molecular mass marker are indicated. Bottom, Processing of Insl6 protein in the presence of furin inhibitor Dec-RVKR-CMK. CHO cells stably expressing c-myc-tagged mInsl6 were cultured in 2% serum and treated with 25 µM furin inhibitor Dec-RVKR-CMK for 24 h (lane 3). Control cells were treated with equal amount of solvent (methanol, lane 2) or without methanol (lane 1). Approximately 20% of the culture medium (from 35-mm culture plate) was subjected to SDS-PAGE size fractionation and Western blot analysis with anti c-myc antibody for the presence of the Insl6 protein. The predicted compositions of the various Insl6 bands and the positions of the molecular mass marker are indicated.

View larger version (43K): [in this window] [in a new window]   FIG. 8. Presence of disulfide bonds in Insl6. A, Intramolecular disulfide bonds in mouse Insl6. Whole-cell lysates (top) or cell culture supernatant (bottom) of CHO cells stably expressing c-myc-tagged mInsl6 protein (mInsl6-CHO; lanes 1–6, top; and lanes 2–5, bottom) or untransfected CHO cells (bottom, lanes 1 and 6) were size-fractionated by SDS-PAGE electrophoresis under either nonreducing conditions (top, lane 1, nonboiled, lane 2, boiled; bottom, lanes 1 and 2, nonboiled) or reducing conditions (dithiothreitol, 1–100 mM, lanes 3–6, top and bottom). The electrophoretic mobility of the Insl6 protein was analyzed by Western blot analysis using anti c-myc antibody. The predicted compositions of the various Insl6 bands and the positions of the molecular mass marker are indicated. B, Differences in disulfide bond formation between mouse and human Insl6 proteins. Cell culture supernatant of CHO cells expressing c-myc-tagged human (lanes 1 and 3) or mInsl6 protein (lanes 2 and 4) were size-fractionated by SDS-PAGE electrophoresis under either nonreducing conditions (lane 1 and 2) or reducing conditions (100 mM dithiothreitol, lanes 3 and 4). The electrophoretic mobility of the Insl6 protein was analyzed by Western blot analysis using anti c-myc antibody. The predicted compositions of the various Insl6 bands and the positions of the molecular mass marker are indicated.

Differential N-linked glycosylation of murine and human Insl6 proteins.
An in silico analysis of the primary structure of the human and murine Insl6 protein sequence using the NetNGly 1.0 software program (http://www.cbs.dtu.dk/services) predicted the presence of a single N-linked glycosylation site (at residue 49 in the human and residue 158 in the mouse). This prediction was tested by analyzing the effect of PNGase F on the electrophoretic mobility of recombinant Insl6 proteins in cell lysate. The electrophoretic mobility of the murine prepro/pro-Insl6 protein in cell lysates was unaffected (Fig. 9A, bottom). In contrast, the electrophoretic mobility of the human prepro/pro-Insl6 peptide was increased by treatment of cell lysates with PNGase F, suggesting that the human protein is modified by N-linked glycosylation (Fig. 9A, upper). However, we were unable to confirm that the secreted hInsl6 peptide is also glycosylated. This is because the predicted glycosylation site on the hInsl6 peptide is located on the putative B peptide, and as detailed in the prior section, we were unable to detect the presence of the B peptide in the secreted hInsl6 peptide. To exclude the possibility that CHO is not an appropriate model system to investigate the glycosylation status of Insl6, we subjected Insl6 ectopically expressed in GC2 to similar analysis. These results (Fig. 9B) verified the presence of N-linked glycosylation in human prepro- or pro-Insl6 and its absence in the murine protein. To further characterize the nature of the glycosylation of the human Insl6 precursor, we tested the effect of Endo H on recombinant human Insl6 protein. The human Insl6 protein was susceptible to Endo H treatment, indicating the presence of immature high mannose residues (Fig. 9C). We confirmed the lack of effect of Endo F on mouse Insl6 by examining mouse testis extracts (Fig. 9D, top). To ascertain the specificity of the anti-Insl6 antibody used to detect endogenous Insl6 in mouse testes, we tested two anti-mInsl6 peptide antibodies, custom-made EE antibody directed against the C chain (Fig. 9D, top) and the commercially available anti-A chain antibody (Phoenix Pharmaceuticals; Fig. 9D, bottom). Western blot analysis of intact testes and primary germ cell extracts with either of these antibodies revealed the presence of a 24-kDa doublet band compatible with prepro- or pro-Insl6 protein. In contrast, a specific signal was not detected in liver extracts. In addition to the 24-kDa doublet, bands that migrated at 15 and 30 kDa were observed when testis extracts were blotted with the EE antibody. The identity of these bands is not clear at the present time. The 15-kDa band could represent a B-C chain intermediate form of Insl6. The 30-kDa band is possibly nonspecific because this band was not observed in germ cell extracts blotted with the anti-A peptide antibody.

View larger version (40K): [in this window] [in a new window]   FIG. 9. Differential N-linked glycosylation of murine and human Insl6 proteins. A, Top, whole-cell lysates of CHO cells stably expressing c-myc-tagged human Insl6 protein were exposed to PNGase F enzyme (+) or vehicle (–), and the products were size-fractionated by SDS-PAGE electrophoresis under reducing conditions and characterized by Western blot analysis using anti c-myc antibody. The positions of the specific bands (1, 1a, 2, and 2a) and the molecular mass markers are indicated. Bands 1a and 2a possibly represent deglycosylated forms of bands 1 and 2, respectively. Bottom, Whole-cell lysates of CHO cells stably expressing c-myc-tagged mInsl6 protein were exposed to PNGase F enzyme (+) or vehicle (–), and the products were size-fractionated by SDS-PAGE electrophoresis under reducing conditions and characterized by Western blot analysis using anti c-myc antibody. The positions of the molecular mass markers are indicated. B, Protein extracts of CHO cells or Insl6-adenovirus (Ad-mInsl6)-infected GC2 cells were exposed to PNGase F enzyme (+) or vehicle (–), and the products were size-fractionated by SDS-PAGE electrophoresis under reducing conditions and characterized by Western blot analysis using anti c-myc antibody. Lanes 1 and 2, Naïve CHO cells; lanes 3 and 4, CHO cells stably expressing hInsl6; lanes 5 and 6, GC2 cells expressing adenovirally mediated mInsl6; lanes 7 and 8, naïve GC2. The cell extracts were treated with PNGase F in certain instances (lanes 2, 4, 6, and 8). The positions of the molecular mass markers are indicated. The results are representative of three independent experiments. C, Whole-cell lysates of CHO cells stably expressing c-myc-tagged hInsl6 protein were exposed to Endo H enzyme (+) or vehicle (–), and the products were size-fractionated by SDS-PAGE electrophoresis under reducing conditions and characterized by Western blot analysis using anti c-myc antibody. The position of the molecular mass marker is indicated. D, Top, aliquots of mouse testis or liver whole-cell extracts were exposed to PNGase F enzyme (+) or vehicle (–), and the products were size-fractionated by SDS-PAGE electrophoresis under reducing conditions and characterized by Western blot analysis using anti-Insl6 antibody (EE). The positions of the molecular mass markers are indicated. Bottom, Aliquots of whole-cell extracts of either mouse germ cells (lane 1) or liver (lane 2) were size-fractionated by SDS-PAGE electrophoresis under reducing conditions and characterized by Western blot analysis using anti-Insl6 A peptide antibody (Phoenix Pharmaceuticals). The position of the molecular mass marker is indicated.

View larger version (44K): [in this window] [in a new window]   FIG. 10. Insl6 is an ubiquinated protein. A, In vitro ubiquination of mouse Insl6. Coupled in vitro transcription-translation reaction using wheat germ extract (lane 1) or reticulocyte lysate (lanes 2 and 3) was used to synthesize [35S] cysteine-labeled mInsl6 in the absence (–) or presence (+) of His-tagged ubiquitin. The products were size fractionated via SDS-PAGE electrophoresis and visualized by autoradiography. The position of putatively ubiquinated higher molecular mass products are indicated in both the reaction without () and with () His-ubiquitin. Note the apparent increase in molecular mass of the bands in the His-ubiquitin reactions. B, Degradation of mInsl6 in vitro is ATP dependent. mInsl6 protein was expressed in a transcription-translation-coupled system where either ATP (+) or ATP-depleting mixture (–) was added 30 min after initiation of the reaction. Aliquots of the reaction at the indicated time-periods were analyzed by SDS-PAGE and autoradiography. C, Degradation of Insl6 is blocked by proteasome inhibitors in vivo. CHO cells stably expressing c-myc-tagged mInsl6 were either exposed to vehicle (–) or 5 µM clasto-lactacystin-ß-lactone (CL) (+), and cells harvested after 4 (lanes 1 and 2) or 24 (lanes 3 and 4) h. The cell lysates were analyzed by Western blot using anti-myc antibody. D, Degradation of Insl6 is blocked by proteasome inhibitors in vitro. Coupled in vitro transcription/translation of Insl6 using reticulocyte lysates performed for the indicated time (hours) in the presence of either vehicle (–) or 5 µM clasto-lactacystin-ß-lactone (Clasto-L) (+). The reaction products were size-fractionated via SDS-PAGE and autoradiographed. E, Stability of Insl6 in the presence of proteasomal or lysosomal inhibitor. CHO cells stably expressing c-myc-tagged mInsl6 were exposed to vehicle, proteasomal inhibitor [clasto-lactocysteine (CL), 10 µM], lysosomal inhibitors [0.2 mM chloroquine (CQ) or 1 µM bafilomycin (Baf)]. mInsl6 protein expressed in these cells was pulse labeled using [35S] cysteine and chased with unlabeled cysteine. At the indicated time points, cells were harvested, immunoprecipitated with anti-myc (9E10) monoclonal antibody, and analyzed by SDS-PAGE as described in Materials and Methods. For a graphical representation of the results, the intensity of the specific bands was quantified by densitometry, and the results were depicted as relative to intensity of the band at time 0 designated as 100%. The x-axis is depicted in log and the y-axis in linear scale.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
Insl6 is a recently identified member of the insulin/relaxin gene superfamily whose biological role is yet to be defined (13, 14, 15). The goal for the present study was to gain insights into the biological actions of Insl6. The main findings of the current study are that Insl6 is a secreted protein expressed in the germ cells of the testis; Insl6 undergoes posttranslational modifications including prohormone processing, disulfide bond formation, N-linked glycosylation, and ubiquitination; and Insl6 is a target for furin action.

Our results indicate that the site of maximal expression of Insl6 is the germ cell of the testis and that expression of Insl6 is restricted to the midpachytene spermatocytes, secondary spermatocytes, and round spermatids stages of germ cell maturation. Although a previous report (15) had also concluded, on the basis of in situ hybridization studies, that Insl6 was predominantly expressed in the germ cell of the testis, Hsu (13) reached the disparate conclusion that Leydig cells are the exclusive site of expression of Insl6. The reason(s) for the discrepancy between the results of the immunohistochemical experiments conducted by Hsu and those of the current study is not clear. Although our results do not exclude the possibility that the Leydig cell may also be a site of expression of Ins6, our results do establish unequivocally that the germ cell is the principal site of Insl6 expression in the adult testis. This conclusion is based on multiple lines of evidence including immunohistochemistry, expression profile of Insl6 mRNA in models of germ cell depletion, and ontogenic profile of mRNA expression in the testis.

Characterized members of the insulin/relaxin superfamily all are secreted proteins with signal peptides in their primary structure and intra- and intermolecular cysteine-linked disulfide bonds in the mature protein. In the present study, we demonstrate that Insl6 is also a secreted protein containing disulfide bonds with intracellular localization to the ER and Golgi. Secretion of Insl6 was confirmed in both CHO and GC2 cells. This secretion could be blocked by BFA treatment, suggesting that Insl6 is secreted through a classic secretory pathway. Ectopic expression of Insl6 in either CHO or GC2 cells resulted in two major bands. In cell lysates, the predominant Insl6 band was approximately 28 kDa in size. In contrast in the supernatant, the dominant band was a doublet of 6/8 kDa (putative A peptide) in size under reducing conditions and 12/14 kDa (putative A + B peptide) under nonreducing conditions. Our results establish for the first time that Insl6, like other members of the insulin family, is processed from precursor (28 kDa) to mature protein (14 kDa) and that the mature protein is linked by disulfide bonds. BFA treatment blocked the secretion of Insl6 in both CHO and GC2 cells, suggesting that the 8-kDa Insl6 peptide is processed and secreted and does not result from nonspecific degradation of dead cells.

Mouse prepro-Insl6 has 191 aa. Because this protein was fused with the myc-His tag, the total number of aa for the fused protein is 22,5 and the predicted size of this protein on SDS-PAGE is approximately 25 kDa for prepro-mInsl6 and 22 kDa for pro-mInsl6. Our results indicate that the putative mouse pro-Insl6 migrated as a 28-kDa doublet, 11–14% larger than expected. At the present time, the reason(s) for the Insl6 precursor migrating as a doublet is not clear. We ruled out the possibility of incomplete reduction of disulfide bonds resulting in oxidized and reduced forms of the protein by treating the protein samples with the alkylating agent iodoacetamide before electrophoresis (data not shown). However, this treatment failed to alter the doublet configuration of the mouse pro-Insl6 protein, indicating that the doublet configuration is not consequent to incomplete reduction or reoxidization of SH groups in the peptide. We excluded the possibility of glycosylation of the murine Insl6 (Fig. 9, A, B, and D) as being a reason for the doublet or the discrepancy in size. In addition to posttranslational modifications and incomplete reduction of disulfide bonds, excess positive or negative charged residues can result in aberrant mobility of a protein on SDS-PAGE (23, 24). A Glu-rich region (EEEEE) is present (aa 22–26) in the mouse Insl6 peptide. Glu-rich regions have been shown to interfere with protein mobility on SDS-PAGE, resulting in an artifactual increase in estimation of molecular weight on SDS-PAGE (25), and it is possible that such a phenomenon may be contributing to the slower than predicted mobility of the mouse Insl6 protein on SDS-PAGE. Total aa for the myc-his-tagged human Insl6 is 249 aa, and the size of the human prepro- and pro-Insl6 is predicted to be approximately 28 and 25 kDa, respectively. Our results indicate that the size of the human prepro/pro-Insl6 is approximately 35 kDa before deglycosylation and 32 kDa after deglycosylation. Hence, the size of the human Insl6 is approximately 11% larger than the predicted size. We hypothesize that similar to the mouse protein, this discrepancy in molecular size could be due to excess negative charge of tandem glutamic acid residues located at aa 56–58 and aa 102–103.

The mammalian PC cadre of proteins include furin/PACE, PC1/PC3, PC2, PC4, PACE4, PC5/PC6, PC7/PC8/LPC, SKI/S1P, and NARC-1/PCSK9. CHO cells have been demonstrated to express furin and PC7 and SKI-1 (26, 27, 28), and LoVo cells are deficient in furin but express other PCs such as PC7 and PACE4 (29, 30). Our results with the furin inhibitor Dec-RVKR-CMK and furin-deficient LoVo cells establish that Insl6 is a target for furin action and that furin is one of the PCs responsible for the 6-/8-kDa doublet configuration of the mature protein on gel electrophoresis. Our results also establish the identity of one of the cleavage sites (RKRR, aa 162–165) of the mInsl6 peptide. Experiments using ectopic expression of furin indicate that this site is a target for furin action. Furthermore, differences in the size of the secreted protein after inhibition of furin activity or mutation of this cleavage site predicts that this site must also be a site for action by nonfurin PC(s). It is noteworthy that the molecular weight profiles of proteins synthesized via ectopic expression of Insl6 in GC2 cells were similar to that observed with ectopic expression in CHO cells, suggesting similarity in the Insl6-directed PC activity in these two cell types. The demonstration of the secretory status of Insl6 could provide clues to the cellular targets of Insl6 action. Thus, in addition to possible autocrine actions on the germ cell, the physical proximity of the germ cell to the Sertoli cell renders the Sertoli cell a likely candidate for Insl6 action. In this regard, the demonstration of Insl6 expression in the brain (31) suggests biological actions of Insl6 outside the testis.

Our studies indicate that Insl6 undergoes other posttranslational modifications such as ubiquitination and N-glycosylation in a species-specific manner. Our results establish that the human Insl6 precursor is N-glycosylated, consistent with the in silico prediction. In contrast, despite the presence of a computer-predicted putative N-glycosylation site in the mouse Insl6 gene, we were unable to demonstrate N-glycosylation of the murine Insl6 protein in either CHO or GC2 cells and in mouse testis extracts. The functional significance of this differential species-specific glycosylation status of the Insl6 protein remains to be elucidated. Protein ubiquitination is an important form of covalent modification that regulates various cellular processes including cell division, growth, communication/signaling, movement, and death/apoptosis (32). The ubiquitin system in the testis plays a role in sperm cell differentiation and the control of cell cycle during spermatogenesis, and mutations in components of the ubiquitin system result in male infertility (33). The ubiquitin proteolytic system has generally been regarded as a cytosolic or nuclear pathway. However, recent studies have identified the presence of ubiquitin and ubiquitin-activating enzyme associated with a post-ER/pre-Golgi compartment with the intracellular degradation of certain secreted proteins (e.g. preproparathyroid hormone-related protein) being dependent on ubiquination. It is further postulated that ubiquination plays a role in ER quality control of misfolded molecules during the synthesis of these proteins (19, 34). A single protein can be modified on one or more lysines with a limited number (<4) of ubiquitin molecules (monoubiquination) or with lysine-linked chains of ubiquitin (polyubiquination) (35, 36). Although polyubiquinated proteins are targeted for degradation via the proteasome, monoubiquinated proteins are preferentially degraded in the lysosome. With respect to Insl6, inhibition of either the proteasomal pathway or lysosomal enzymatic activity resulted in prolongation of the half-life of the protein, although the effect of lysosomal inhibition was more dramatic and complete. How can the half-life of the Insl6 protein be apparently affected by perturbation of both the proteasome and the lysosomal pathways? We propose that Insl6 is monoubiquinated and that the primary mode of degradation is by the lysosomal pathway. We hypothesize that the partial effect of proteasome inhibitors on Insl6 degradation is the indirect result of lower cellular levels of ubiquitin that can be induced by proteasome inhibitors (37, 38) and does not reflect a direct role of the proteasome in Insl6 degradation. A caveat in our results is that our results regarding ubiquination of Insl6 were obtained exclusively using an in vitro system. Further studies will be required to confirm the presence, characteristics, and biological role of in vivo ubiquination of the Insl6 protein.

In conclusion, our results indicate that Insl6 is expressed in the germ cells of the testis. Data presented in the current study demonstrate that Insl6 is a disulfide-linked secreted protein using the classic secretion pathway that undergoes processing from preprohormone to mature hormone, with furin playing a role in this processing. Insl6 is also subjected to ubiquitination and N-glycosylation in a species-specific manner.

View larger version (34K): [in this window] [in a new window]   FIG. 11. Mouse Insl6 protein. The predicted signal peptide (italicized), B chain (boxed), C chain (gray), and A chain (boxed) are specified. The putative locations of the disulfide bonds and furin cleavage site are indicated.

	Acknowledgments

	Footnotes

 
This work was supported by the National Institutes of Health [Grants HD HD044436 and DK49845 (to R.K.M.) and T32DK07729], by the Children’s Hospital of Pittsburgh (Renzeihausen Fund), and by the Vira I. Heinz Foundation. This work was also supported in part by the Michigan Diabetes Research and Training Center Grant (P60DK-20572) from the National Institute of Diabetes and Digestive and Kidney Diseases.

The authors have nothing to disclose.

First Published Online August 24, 2006

Abbreviations: aa, Amino acid; BFA, brefeldin A; CI, cytotoxicity index; C_T, threshold cycle; Endo, endoglycosidase; ER, endoplasmic reticulum; FAM, 5-carboxyfluorescein; FBS, fetal bovine serum; FITC, fluorescein isothiocyanate; HRP, horseradish peroxidase; ISL, insulin-like peptide; PC, prohormone convertase; PNGase F, peptide N-glycosidase F.

Received April 19, 2006.

Accepted for publication August 14, 2006.

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