LYZL6, an acidic, bacteriolytic, human sperm-related protein, plays a role in fertilization

Abstract

Lysozyme-like proteins (LYZLs) belong to the c-type lysozyme/α-lactalbumin family and are selectively expressed in the mammalian male reproductive tract. Two members, human sperm lysozyme-like protein (SLLP) -1 and mouse LYZL4, have been reported to contribute to fertilization but show no bacteriolytic activity. Here, we focused on the possible contribution of LYZL6 to immunity and fertilization. In humans, LYZL6 was selectively expressed by the testis and epididymis and became concentrated on spermatozoa. Native LYZL6 isolated from sperm extracts exhibited bacteriolytic activity against Micrococcus lysodeikticus. Recombinant LYZL6 (rLYZL6) reached its peak activity at pH 5.6 and 15 mM of Na+, and could inhibit the growth of Gram-positive, but not Gram-negative bacteria. Nevertheless, the bacteriolytic activity of rLYZL6 proved to be much lower than that of human lysozyme under physiological conditions. Immunodetection with a specific antiserum localized the LYZL6 protein on the postacrosomal membrane of mature spermatozoa. Immunoneutralization of LYZL6 significantly decreased the numbers of human spermatozoa fused with zona-free hamster eggs in a dose-dependent manner in vitro. Thus, we report here for the first time that LYZL6, an acidic, bacteriolytic and human sperm-related protein, is likely important for fertilization but not for the innate immunity of the male reproductive tract.

Fig 1. Tissue expression patterns of LYZL6.

A) RT–PCR analysis. Lane M: DL2000 DNA marker. The expected sizes of PCR products were 296 bp for LYZL6 and 247 bp for G3PDH. Totally 14 tissues were examined. Nine of them were shown and the rest included the pancreas, small intestine, colon, thymus and skeletal muscle. B) Anti-LYZL6 serum specificity analysis. Top panel: SDS-PAGE; Bottom panel: western blotting. Lane M: molecular weight (MW) marker; The expected MW was ~14.7 kDa for LYZ and ~32.0 kDa for His-tagged LYZL6 (HLYZL6) and His-tagged LYZL4 (HLYZL4), which the extra MW resulted from Trx-tag, S-tag and restriction enzyme site of the pET32a vector. The amount of His-tagged LYZL6 and His-tagged LYZL4 loaded was 0.5 μg and that of LYZ was 0.1 μg. C) Western blot analysis of human tissue extracts. A band corresponding to full-length LYZL6 was evident in the testis and epididymis. No expression was detected in the other human tissues. D) Western blot analysis of LYZL6 in human semen. LYZL6 was identified in protein lysates representing different numbers of ejaculated spermatozoa (left), but not in seminal plasma (right). β-actin and LYZ were used as internal controls respectively.

Fig 2. Isolation of native LYZL6 from human sperm extracts.

A) Elution profile of chitin affinity chromatography. Inset: SDS–PAGE of the chitin column peak. Lane M: MW marker; Lane 1: peak 1. B) Elution profile of anion exchange chromatography. Inset: SDS–PAGE of the HiTrap Q column peaks. Lane M: MW marker; Lane 1–3: peak 1, 2, and 3. Bands in the gel were identified by MS.

Fig 3. Bacteriolytic activity of LYZL6.

A) Homology modeling of LYZL6 and LYZ. The structures of LYZL6 and LYZ were predicted using MODELLER 9.5. The active centers (AC) of LYZL6 and LYZ were shown (arrows). B) LYZL6 displayed bacteriolytic activity specifically against M. lysodeikticus but not E. coli. Bacteriolytic activity of LYZL6 was assessed using radial diffusion assay. Native LYZL6 in PBS (pH 6.0) was added to wells and the zone of clearance was observed using M. lysodeikticus (arrow), but not using E. coli as substrate. LYZ was used as a positive control and PBS was used as a negative control.

Fig 4. Identification of rLYZL6 expressed in P. pastoris.

A) SDS–PAGE of LYZL6 expression at various time points after methanol induction. Lane M: MW marker. Supernatant protein (~10 μg per lane) was loaded. B) Western blot analysis of the fermentation supernatant. Lane M: MW marker; Lane 1–2: fermentation supernatant from un-transformed yeast; Lane 3–4: fermentation supernatant from recombinant yeast. C) SDS–PAGE of fractions collected by chitin affinity chromatography. The amount of rLYZL6 loaded was 0.1 μg (lane 1), 0.5 μg (lane 2), 1 μg (lane 3), 2 μg (lane 4–6). D) Silver stained SDS–PAGE of samples collected through size exclusion chromatography steps. Lane M: MW marker; Lane 1–3: rLYZL6 (~0.5 μg per lane). The samples were obtained from three separate experiments.

Fig 5. Effect of pH and ion concentration on the activity of rLYZL6.

A) Activities of rLYZL6 and LYZ at different pH values, with a maximum activity at pH ~5.6. B) Effect of Na⁺ ion concentration on activities of rLYZL6 and LYZ. For all of the assays, rLYZL6 solution was added to substrate suspension and identical amounts of human milk LYZ were used as controls. All values have been normalized to the peak activity for each curve (100%).

Fig 6. Bacteriolytic activities of rLYZL6 or LYZ under different conditions.

The results are presented as the percentage of the control incubated without protein. The rLYZL6 protein showed high bacteriolytic activities against M. lysodeikticus, and B. subtilis. The growth inhibition caused by rLYZL6 decreased significantly after transferring to a high pH (P < 0.001). The rLYZL6 protein exhibited weaker bacteriolytic activities than LYZ against the sensitive strains (P < 0.05). The letters (a and b) were used to indicate the comparisons.

Fig 7. Immunofluorescence staining of LYZL6 in spermatozoa and rLYZL6 bound to zona-free hamster eggs.

A) Live and permeabilized spermatozoa were incubated with the anti-LYZL6 serum or preimmune rabbit serum, followed by a rhodamine-conjugated goat anti-rabbit secondary antibody. A band-like pattern (red) was detected on the postacrosomal region for LYZL6 in acrosome-reacted (AR) and in acrosome intact (AI) cells. No similar staining of LYZL6 was seen in the control cells. The FITC-PSA staining was used to distinguish membrane changes in spermatozoa due to acrosome reaction. B) The eggs were incubated with rLYZL6 (upper panels) or without rLYZL6 (lower panels) and probed with the anti-LYZL6 serum. Scale bar = 50 μm.

Fig 8. Effect of LYZL6 antiserum on sperm-egg binding and fusion.

A) The mean number (± SD) of spermatozoa bound per egg did not differ significantly between the control and the antiserum-treated groups (control: 29 ± 3.07; anti-LYZL6 serum: 1: 100, 23 ± 2.48; 1: 200, 26 ± 3.20; 1: 400, 28 ± 3.56; 1: 800, 27 ± 2.34; 1: 1600, 30 ± 3.85). B) The mean number of spermatozoa fused per egg decreased significantly following incubation of spermatozoa with higher antiserum concentrations (control: 4.68 ± 0.69; anti-LYZL6 serum: 1: 100, 1.16 ± 0.51; 1: 200, 1.54 ± 0.36; 1: 400, 2.77 ± 0.46; 1: 800, 3.98 ± 0.73; 1: 1600, 4.35 ± 0.59). *P < 0.05; **P < 0.01.