Fanibunda, Sashaina E MSc; Velhal, Shilpa M BSc; Raghavan, Vijaya P PhD; Bandivdekar, Atmaram H PhD
HIV is known to be transmitted primarily through the sexual route.1 Initially, cell-free virus and seminal leukocytes were considered to be the sole source responsible for the sexual transmission of HIV, as sexually transmitted infections are often associated with high seminal leukocytes, and sexually transmitted infection is one of the risk factors associated with HIV infection. However, subjects with low seminal leukocyte count or absence of sexually transmitted infection are also known to acquire HIV infection. Moreover, studies using monkey models indicated that due to the acidic pH of the vagina and the temporal sequel of seminal leukocytes in the female vaginal tract, infection of female through cell-free virus or by seminal leukocytes from the male seems less likely. Furthermore, the viral load required for transmission of HIV through the vaginal route has been demonstrated to be very high as compared with that of the systemic route.2 This suggests that spermatozoa may also be responsible for the sexual transmission of HIV. The powerful technique of atomic force microscopy has been employed to examine the localization of HIV on the spermatozoa of HIV-infected patients. Viral particles were located on the outer membrane surface of spermatozoa, and the merging of such particles on the surface of the spermatozoa was detected.3 Studies by Bagasra et al4,5 have localized HIV DNA in the ejaculated spermatozoa of infected individuals by in situ polymerase chain reaction (PCR). HIV proviral DNA has also been detected in germ cells at all stages of differentiation, in the testes of HIV-seropositive men.6-8 Electron microscopic studies have revealed HIV bound to spermatozoa of seronegative donors incubated in vitro with cell-free virus, followed by subsequent transfer of HIV to leukocytes in culture,9-11 through the entry of entire sperm head into the human leukocyte antigen-DR-positive leukocyte cells.10 It has also been reported that HIV-infected spermatozoa have the ability to fertilize oocytes and transfer the virus into the resulting embryo, but cell-free virus is not able to bind or penetrate the oocyte in vitro.12 These studies provide strong evidence of HIV binding to spermatozoa and further transmission of HIV to urogenital cells, via spermatozoa. However, the lack of conventional CD4 receptors on germ cells13 and on mature spermatozoa14 suggests the presence of an alternate receptor for HIV on spermatozoa. In an attempt to understand the CD4-independent mechanism of HIV binding to sperm, a 160kDa HIV-binding sperm protein has been identified for the first time by our group.15 Both gp120 HIV env glycoprotein and cell-free virus bind specifically to the 160kDa sperm protein. This 160kDa HIV-binding protein was found to be different from the conventional CD4 receptor for HIV. The present communication further characterizes the CD4-independent 160kDa HIV-binding protein and reports that this protein is the human mannose receptor.
Preparation of Sperm Protein Extract
Semen samples used for extraction of sperm proteins were obtained from a local infertility clinic and also from healthy fertile volunteers. Samples were analyzed in accordance with World Health Organization Guidelines,16 and only normozoospermic samples (count > 20 million/mL, progressive motility > 50%, morphology > 30% normal forms) were included in the study. Samples with leukocyte count greater than 4-6/per high power field (greater than 1 million/mL) were excluded. The semen samples were washed thrice with HAM's F10 medium (Hi Media, Mumbai, India) containing 0.2% bovine serum albumin (BSA), and leukocyte-free sperm were separated from seminal plasma by the swim-up technique as described earlier.15 Spermatozoa were pooled; solubilized in lysis buffer containing 10 mM Tris, pH 8.0, 1% Triton X-100, and 10-μL/mL cocktail of protease inhibitors (Sigma Chemical Company, St Louis, MO); and centrifuged at 1800 g, for 15 minutes at 4°C. Supernatants were recovered, dialyzed extensively against 10 mM ammonium carbonate, lyophilized, and stored at −80°C until use. Protein concentration was estimated by Peterson's modification of Folin-Lowry method17 using BSA as a standard.
Partial Purification of 160kDa CD4-Independent HIV-Binding Protein
The 160kDa CD4-independent HIV-binding protein was partially purified from sperm protein extract by ion exchange chromatography using fast protein liquid chromatography on a Mono Q HR 10/10 column (Pharmacia). The sperm protein extract was dissolved in 0.01 M Tris-HCl, pH 7.0 (initial buffer), equilibrated by dialysis with the same buffer and fractionated on a Mono Q HR 10/10 column preequilibrated with the initial buffer. After elution of unbound proteins with the initial buffer for 10 minutes, the bound proteins were eluted from the column using a gradient of 0-1 M NaCl in 0.01 M Tris-HCl, pH 7.0, at the flow rate of 1 mL/min. The HIV-binding protein fraction was identified by Western blot analysis using gp120 HIV env glycoprotein and its antibody as described earlier.15
Partial Amino Acid Sequencing of 160kDa CD4-Independent HIV-Binding Protein
The partially purified fraction containing the gp120 reacting 160-kDa protein was subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis and electrophoretically transferred onto a polyvinylidene fluoride membrane, using 10 mM 3-(cyclohexylamino)-1-propane sulphonic acid buffer, pH 11.0, containing 10% methanol. The gp120 reacting 160kDa protein band was visualized by staining with 0.1% Ponceau S in 1% acetic acid for 5 minutes, followed by destaining with 5% acetic acid. After several washes with double-distilled water, the gp120 reacting 160kDa protein band was excised and subjected to N-terminal amino acid sequencing by automated Edman degradation at the Protein/DNA Technology Center, Rockefeller University, New York, NY. The PVDF-bound protein was then further subjected to tryptic digestion, and the peptides generated were purified by microbore reverse-phase chromatography on Hewlett-Packard 1090 HPLC system. The partial N-terminal sequence of the major peptide thus obtained was determined similarly.
Amplification of Corresponding cDNA Encoding the 160kDa Protein by Mixed Oligonucleotide-Primed Amplification of cDNA
Based on the partial amino acid sequence of the N-terminal peptide and the second major peptide obtained by tryptic digestion of the protein, degenerate deoxyinosine containing primers were designed; sense primer corresponding to the N-terminal peptide 5′-CTICTGGTITTTGCCTCTGTC-3′ and antisense primer corresponding to the second peptide 5′-IACICCGCTGTTGAAGCTCAG-3′ (I = inosine base).
These were used for mixed oligonucleotide-primed amplification of cDNA(MOPAC), using human testicular cDNA library as template. Human testicular cDNA library (3 × 108 clones/mL), diluted 1:10, was amplified using 0.4 μM of each primer, 200 μM dNTPs, 3U of Taq polymerase (Bangalore Genei, Bangalore, India), in a 25-μL reaction. Amplification was carried out for 35 cycles with each cycle comprising denaturation at 94°C for 30 seconds, annealing at 60°C for 45 seconds, and extension at 72°C for 1 minute. The products were analyzed on a 1.2% agarose gel, stained with ethidium bromide, and visualized under UV transillumination. The negative control did not include the template, and a second negative control did not include Taq DNA polymerase. The PCR product was gel eluted, cloned into TOPO vector (Invitrogen), and sequenced followed by in silico analysis of the sequence obtained.
PCR Amplification and Sequencing of Mannose Receptor cDNA From Human Testis
To obtain the sequence of mannose receptor from testis, cDNA encoding the mannose receptor protein was isolated by PCR amplification of human testicular cDNA using human mannose receptor-specific primers. Human testicular Marathon Ready cDNA (Clontech, Palo Alto, CA) was used as template and amplified by using a High-Fidelity Advantage cDNA PCR kit (Clontech) and a primer set of sense 5′-CCATGAGGCTACCCCTGCTCCTGGTT-3′ and antisense 5′-CTAGATGACCGAGTGTTCATTCTG-3′ (primers were commercially synthesized by Life Technologies) with a PCR program of 1 cycle of 94°C for 30 seconds, 35 cycles each of 94°C for 5 seconds and 68°C for 4 minutes, and 1 cycle of 68°C for 7 minutes. The products were analyzed on a 1.2% agarose gel and visualized under UV transillumination. The amplified DNA product was then gel eluted and sequenced by primer walking (Bangalore Genei).
Detection of Mannose Receptor in Sperm Extract by Western Blot
To further determine the identity of the 160kDa HIV-binding protein as mannose receptor, we determined the size of mannose receptor on human spermatozoa. Western blot of sperm extract was performed using antibodies against mannose receptor. In brief, sperm protein extract (75 μg) was resolved by SDS-PAGE on a 6% polyacrylamide gel and electrophoretically transferred onto a nitrocellulose (NC) membrane. The NC membrane was sequentially incubated for 1 hour at room temperature with 5% nonfat dried milk in 1× TBS (Tris-buffered saline: 100 mM Tris-HCl, pH 7.5, 150 mM NaCl) and then overnight at 4°C with monoclonal antibody to hMR (Clone 19.2, Pharmingen) diluted 1:250 in 1× TBS containing 5% nonfat milk. After 3 washes with 1× TBS containing 0.1% Tween 20 (TBS-T), the NC membrane was incubated for 1 hour at room temperature in sheep anti-mouse secondary antibody conjugated with horseradish peroxidase diluted 1:1000 in 1× TBS containing 5% nonfat milk. The NC membrane was washed thrice with 1× TBS-T and visualized using the chemiluminescence detection system (Amersham) on Kodak X-Omat XK-5 film. Western blot analysis of the same sample was also performed using gp120 env glycoprotein and its antibody as a probe, as described earlier.15 The binding of antibodies to mannose receptor and of gp120 to the 160kDa protein were compared.
Fluorescent Localization of HIV gp120-Binding Sites and Mannose Receptor on Human Spermatozoa
Purified (recombinant) HIV gp120 from the IIIB isolate conjugated to fluorescein isothio cyanate (FITC) was obtained from Immunodiagnostics (Woburn, MA, USA). Human mannose receptor antibody (clone 19.2) FITC conjugated (hMR-FITC) or control IgG antibody FITC labeled (IgG-FITC) was obtained from Pharmingen (San Diego, CA). Three sperm samples from proven fertile healthy donors were processed. A swim-up preparation of human spermatozoa was used-briefly, spermatozoa were washed free of seminal plasma and pelleted, the pellet was overlaid with Ham's F10 and 3.5% human serum albumin for 45 minutes at 37°C, and medium containing motile sperm was carefully withdrawn. Spermatozoa were studied immediately after swim-up or after capacitation to observe potential changes in the gp120-binding patterns. Spermatozoa were capacitated by incubation in Ham's F10 medium supplemented with 3.5% human serum albumin for 7 hours at 37°C in 5% CO2. Spermatozoa were centrifuged out of the medium, washed twice in MgCl2/N-2-Hydroxyethyl piperazine-N′-2-ethanesulfonic acid (HEPES) buffer supplemented with 20 mM CaCl2 (Ca buffer), and smeared onto glass slides. The slides were air dried, fixed in chilled acetone for 20 minutes, and frozen at −20°C till use. To prevent nonspecific binding, the smears were blocked using 1% BSA, followed by incubation with gp120-FITC diluted 1:250 in Ca buffer, or with hMR antibody FITC labeled, at 4°C overnight. Subsequently, the slides were washed twice in MgCl2/HEPES buffer. Nuclei were counterstained using propidium iodide and mounted in Vectashield (Vector Laboratories, Cambridgeshire, United Kingdom) antiquenching mounting medium. The edges of the coverslips were sealed with nail varnish, and the slides were stored in the dark at −20°C up to 2 days before examination. Spermatozoa incubated with FITC-conjugated protein (BSA) (Sigma) served as a negative control in gp120-binding studies, whereas control spermatozoa were labeled with isotope-matched IgG antibody FITC conjugated in hMR localization studies. All experiments were repeated in triplicates (n = 3). To determine the specificity of gp120-FITC binding to sperm, spermatozoa were incubated with molar excess of unlabeled gp120 in the presence of labeled gp120. Further, inhibition experiments were performed with an hMR ligand antagonist yeast mannan. Capacitated spermatozoa were incubated with 10-fold (0.05 mg/mL) or molar excess (5 mg/mL) of mannan along with gp120-FITC. Confocal images were obtained using the Zeiss LSM-510 Meta confocal laser scanning microscope (Figure 7).
Further, the binding of gp120 to spermatozoa was tracked over a time course of 24 hours. Swim-up spermatozoa were labeled with gp120-FITC conjugate for varying time intervals of 0, 1, 2, 4, 12, 16, and 24 hours. Briefly, after swim-up, spermatozoa were washed in Ca buffer and incubated with gp120-FITC at 1:250 dilution, at 37°C for varying time intervals as outlined above. Spermatozoa were then washed twice in MgCl2/HEPES buffer, nuclei were counterstained using propidium iodide and mounted in Vectashield antifade mountant. Detection of external and internal gp120 was done by Z-stacking using a Zeiss LSM-510 Meta laser scanning confocal microscope. Ten spermatozoa were analyzed at each time point studied.
About 25 mg of total sperm protein was extracted from 150 sperm samples, and about 1 mg of partially purified HIV-binding protein was obtained after ion exchange chromatography. Figure 1 shows the elution profile of Triton X-100-extracted human sperm proteins after ion exchange chromatography. The shaded fraction contains the gp120-binding protein as detected by Western blot analysis. To determine the partial amino acid sequence, the 160kDa protein-containing fraction was electrophoretically transferred onto a PVDF membrane, and the gp120 reacting band was excised and sequenced. The following is the partial N-terminal amino acid sequence of the 160kDa HIV-binding protein and its peptide obtained by tryptic digestion.
Partial N-terminal amino acid sequence of 160kDa HIV-binding sperm protein:
* X R L K L L L V F A S V
Partial N-terminal amino acid sequence of peptide obtained by tryptic digestion of 160kDa HIV-binding sperm protein:
X L S L S F N S G V E
The partial amino acid sequences showed identity with human mannose receptor as shown below:
X R L K L L L V F A S V Partial N-terminal sequence of 160kDa
M : : P : : : : : : : : Human Mannose receptor
X L S L S F N S G V E Partial N-terminal sequence of the peptide of 160kDa
L N : : : : : : : W Q Human Mannose receptor
: Identical amino acid residue as in the upper line.
In silico analysis of the partial amino acid sequence of the 2 peptides was performed using the short nearly exact matches tool of the National Center for Biotechnology Information database. The N-terminal amino acid sequence of 160kDa sperm protein exhibited 83.3% identity with the human macrophage mannose receptor (accession no. P22897), whereas the peptide obtained by tryptic digestion of the protein demonstrated 63.6% identity to hMR.
Corresponding cDNA Sequence Encoding the 160kDa Protein Obtained by Mixed Oligonucleotide-Primed Amplification of cDNA
A product of 850 base pair (bp) was obtained (Fig. 2) by PCR, using human testicular cDNA library as template and degenerate deoxyinosine containing primers corresponding to the 2 peptides of the 160kDa protein. (Peptide sequences are mentioned above.) The cDNA sequence of this 850-bp PCR product exhibited 99% identity with the published human macrophage mannose receptor sequence (accession no. NM_002438.1).
Expression of hMR in Human Testis
PCR was performed using human testicular Marathon ready cDNA as template and specific primers based on human macrophage mannose receptor published sequence and resulted in the amplification of a product of approximately 4 kilobase (kb), which is the estimated size of the coding sequence of mannose receptor gene. Figure 3 depicts the 4-kb PCR product. On sequencing, the product exhibited 99% identity with the macrophage mannose receptor sequence (accession no. NM_002438.1). This sequence was deposited in the GenBank database and was assigned accession no. DQ663787.
The cDNA sequence of the 850-bp PCR product exhibited 99% identity with the corresponding region of the testicular human mannose receptor transcript with accession no. DQ663787.
Mannose Receptor on Human Spermatozoa
The expression of mannose receptor on spermatozoa was evaluated by Western blot analysis, using a monoclonal antibody specific to the mannose receptor. The size of sperm mannose receptor was found to be of 160kDa. Figure 4A depicts the band of 160kDa, corresponding to the mannose receptor. This band was found to be at the same position as the CD4-independent HIV-binding protein (Fig. 4B).
Fluorescent Localization of HIV gp120-Binding Sites and Mannose Receptor on Human Spermatozoa
To localize HIV gp120-binding sites on human spermatozoa, gp120-FITC was used. In swim-up spermatozoa, 2 main fluorescent patterns were observed, as demonstrated in Figure 5A. One covering the entire acrosomal area (cap pattern) and the other with the label concentrated at the equatorial segment (bar pattern). Cap pattern was observed in about 1% of spermatozoa. Bar pattern was observed in approximately 10% of spermatozoa after swim-up and increased to approximately 90% after incubation in capacitating medium (Fig. 5C). No staining was observed in control spermatozoa incubated with FITC-BSA (Figs. 5B, D). A similar pattern of staining was observed, when hMR was localized on human spermatozoa (Fig. 6); cap pattern was observed in about 1% of spermatozoa, whereas bar pattern was observed in approximately 10% of ejaculated sperm, which increased to approximately 90% after capacitation.
Spermatozoa incubated with molar excess of unlabeled gp120 in the presence of gp120-FITC did not exhibit staining (Fig. 7B), demonstrating the specificity of gp120-FITC binding to sperm. In competition experiments, mannan was used at 10-fold and molar excess of gp120. Mannan reduced the binding of gp120 to the sperm surface, and complete inhibition of gp120 binding was observed at molar excess concentrations (Fig. 7C).
In the gp120-tracking experiment, spermatozoa were examined for surface gp120 and internalization at different time points 0, 1, 2, 4, 12, 16, and 24 hours. At 0 hour, there was no binding of gp120-FITC to spermatozoa. Z-stack of the fluorescent pattern observed in 10 individual spermatozoa was performed at each time point. No internalization of gp120 was observed at any of the time points studied, with surface labeling evident at each time point. The gp120 remained bound to mannose receptor at the surface only, over the entire 24-hour duration. Representative confocal micrographs of the Z-stack data obtained for the cap pattern and bar pattern of HIV gp120 binding are depicted in Figures 8A, B, respectively.
HIV binds to human spermatozoa in a CD4-independent manner. We have previously reported a CD4-independent molecule of 160kDa on human spermatozoa that exhibits specific binding to cell-free HIV and gp120 env glycoprotein. This 160kDa HIV-binding protein was found to be distinct from the conventional CD4 receptor and it was also found that it is not a glycolipid.15 The presence of HIV has also been detected in testicular germ cells at all stages of spermatogenesis but not in Leydig cells or Sertoli cells of infected individuals. Moreover, testicular germ cells have also been reported to be devoid of the conventional CD4 receptor, suggesting that HIV also binds in a CD4-independent manner to testicular germ cells. The present study further characterizes the 160kDa HIV-binding sperm protein and provides evidence that sperm mannose receptor may function as the 160kDa CD4-independent HIV-binding protein.
The partial amino acid sequence of the 160kDa HIV-binding protein yielded 2 peptides that exhibited identity with the human macrophage mannose receptor; thus we postulated that the 160kDa HIV-binding protein on spermatozoa could also be mannose receptor. The hMR cDNA of 4 kb derived from human testis exhibited 99% sequence identity with human macrophage mannose receptor. The cDNA sequence of the 850-bp PCR product exhibited 99% identity with the corresponding region of the human mannose receptor transcript with accession no. DQ663787 as mentioned above and also with the published human macrophage mannose receptor sequence (accession no. NM_002438.1).
Further, the sequences of the two peptides derived from the 160kDa HIV binding protein, were found to be present in the translated cDNA sequence of full length human testicular mannose receptor. These two peptides were found to be 286 amino acids apart, which corresponds to a size of 850 bp as obtained by PCR. This corroborates that the 160kDa HIV binding sperm protein is indeed human mannose receptor.
The observation that approximately 10% of ejaculated spermatozoa expressed mannose receptor on the surface at the equatorial region is concomitant with the expression of mannose-binding sites on human spermatozoa, in studies by Chen et al.18 The present study also points to the fact that HIV gp120 can bind to only a fraction of ejaculated spermatozoa. Not surprisingly, HIV infection in patients with AIDS is almost always observed in a fraction of ejaculated spermatozoa.9,19 Our results demonstrate that the sperm mannose receptor can serve as a binding site for HIV-1 through the binding of glycosylated gp120 to mannose receptor. Direct involvement of hMR in HIV gp120 binding was further supported by displacement studies using molar excess of cold unlabeled gp120 and competitive binding studies using mannan.
To the best of our knowledge, human mannose receptor as the CD4-independent HIV-binding protein on spermatozoa has not been demonstrated earlier. However, in astrocytes, inhibition assays using mannan and anti-human mannose receptor antiserum demonstrated that HIV-1 infection occurs through a CD4-independent pathway, with mannose receptor functioning as the primary receptor.20 Interestingly, in macrophages, besides CD4 and chemokine coreceptor 5 (CCR5), 60% of HIV binding has been found to occur via mannose receptor.21
The mechanism of HIV binding to mannose receptor is unknown. It is reported that approximately 50% of the 120kDa molecular weight of gp120 is provided by carbohydrate, all of it is N-linked. No O-linked glycosylation has been identified. There are 24 N-linked glycosylation sites in the gp120 amino acid sequence, attached to the amino acid asparagines. These are well conserved and 33% of these are of the high-mannose type.22-24 In sperm, the mannose-binding protein or mannose receptor has been extensively characterized,18,25,26 and it has been demonstrated that the putative mannose receptor on sperm binds to high-mannose oligosaccharides,18,27,28 which are abundantly present on gp120. Furthermore, studies have demonstrated that gp120 binds to mannose-binding lectins (C-type lectins), via the glycan residues; deglycosylation of gp120 abrogates this interaction.29,30 Indeed, the human mannose receptor is a member of the C-type lectin family, and its ligand binding is mediated by mannosylated and/or mannose-rich glycan moieties present in its ligands,31 is further evidence in favor of spermatozoa mannose receptor as the HIV gp120-binding protein.
Over the time course of 24 hours, HIV gp120 did not enter into sperm but remained bound to mannose receptor on the outer surface of sperm. This is not surprising as spermatozoa being highly differentiated cells lack endocytosis machinery and are transcriptionally inactive and do not support active transcription and protein synthesis, hence also excluding a role in HIV replication. Spermatozoa may thus serve as potential carriers of HIV from male to female partners, through HIV gp120 binding to mannose receptor on the sperm surface. Studies by Scofield10 have provided a direct role of spermatozoa in the horizontal transmission of HIV, through HIV binding to sperm surface, followed by spermatozoa binding to HLA-DR-positive cells, directly delivering HIV into these cells32 and also causing activation of HIV replication in HLA-DR-positive cells.33 Further, ejaculated spermatozoa in the female reproductive tract undergo capacitation and thus exhibit increased mannose receptor expression. These spermatozoa may further facilitate the binding of free HIV to sperm in the lower female reproductive tract and further transport of HIV to distal sites in the female reproductive tract.
The current study presents mannose receptor as a potential CD4-independent HIV-binding protein on spermatozoa. Spermatozoa may serve as carriers of HIV, through the binding of HIV gp120 to sperm mannose receptor. However, the role of sperm in HIV transmission, via mannose receptor, merits further investigation.
Protein sequence analysis was provided by The Rockefeller University Protein/DNA Technology Center, which is supported in part by National Institutes of Health-shared instrumentation grants and by funds provided by the US Army and Navy for purchase of equipment. Recombinant HIV-1 IIIB gp120 was supplied by ImmunoDiagnostics Inc and the Centralised Facility for AIDS Reagents supported by EU Programme European Vaccine against AIDS/MRC (contract QLKZ-CT-1999-00609) and the UK Medical Research Council. Antiserum to HIV-1 gp120 #20 (CHO) was provided by Dr S. Ranjbar and the Centralised Facility for AIDS Reagents supported by EU Programme EVA (contract QLK2-CT-1999-00609) and the UK Medical Research Council. We are grateful to Dr C.P. Puri, Former Director, and Dr P. K. Meherji, Officer in Charge, National Institute for Research in Reproductive Health for their encouragement and support in carrying out this study.
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