Human fibrosarcoma HT1080 cells, purchased from Japanese Cancer Research Resources Bank (JCRB), were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Nissui, Tokyo, Japan), supplemented with 10% (v/v) fetal bovine serum, 200 U/ml penicillin G, 200 mg/l kanamycin, 600 mg/l L-glutamine, and 2.25 g/l NaHCO₃ at 37°C in a humidified incubator with 5% CO₂.

The human HYAL1-myc-his₆ gene was amplified from a human prostate cancer LNCaP cell cDNA library and subcloned into the pCI-neo vector (Promega, Madison, WI). The permanent cell line expressing HYAL1-myc-his₆ was established by transfecting the vector into HT1080 cells, followed by 400 μg/ml G418 (Roche Applied Sciences, Indianapolis, IN) selection. The clonal cells that expressed high levels of myc-his₆-tagged HYAL1 were designated HT1080-HYAL1-MH cells. The cells that were transfected with pCI-neo vector were designated HT1080-neo.

To perform western blot analysis, we used a slightly modified version of a previously described methods (20–23). Cells were lysed in a lysis buffer [50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.1% sodium dodecyl sulfate (SDS), 1% Triton X-100, 1% sodium deoxycholate and 1 mM phenylmethylsulfonyl fluoride] at 4°C with sonication. The lysates were centrifuged at 14,000 rpm for 10 min, and the amount of protein was measured by staining with Coomassie Brilliant Blue (CBB) G-250 (Bio-Rad Laboratories, Hercules, CA). Loading buffer (350 mM Tris-HCl, pH 6.8, 30% glycerol, 0.012% bromophenol blue, 6% SDS and 30% 2-mercaptoethanol) was added to each lysate, which was subsequently boiled for 3 min and electrophoresed on SDS-polyacrylamide gels. Proteins were transferred to PVDF membranes and immunoblotted with anti-c-myc (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) or anti-α-tubulin (Sigma, St. Louis, MO) antibodies. Detection was performed with enhanced chemiluminescence reagent (Millipore Corporation, Billerica, MA).

To purify recombinant HYAL1 from the conditioned medium, HT1080-HYAL1-MH cells were cultured in serum-free DMEM for 24 h, and the conditioned media was concentrated on an ultrafiltration membrane and incubated with Ni-NTA agarose (Qiagen, Hilden, Germany) for 2 h at 4°C. The Ni-NTA agarose was washed five times with phosphate-buffered saline (PBS) and eluted with 500 mM imidazole.

To purify recombinant HYAL1 from the whole-cell lysate, HT1080-HYAL1-MH cells were lysed with binding buffer (50 mM Tris-HCl, pH 7.5, 0.5 M NaCl, 8 M Urea, 20 mM imidazole), and cell lysate was incubated with Ni-NTA agarose for 2 h at 4°C. The Ni-NTA agarose was washed four times with PBS and eluted with 500 mM imidazole.

The obtained samples were electrophoresed on SDS-polyacrylamide gels and stained with CBB R-250. The purified proteins were used for mass spectrometry (24,25).

Purified recombinant HYAL1 was subjected to SDS-polyacrylamide gels. After CBB staining, the bands were excised and treated with 0.05 μg of sequencing-grade modified trypsin (Promega) at 37°C for 12 h in 0.1 M Tris-HCl, pH 8.0. The digests were desalted using Zip TipC18μ (Millipore Corporation) and applied to matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS) on a Ultraflex TOF/TOF MS (Bruker Daltonics, Bremen, Germany) in reflector mode using α-cyano-4-hydroxycinnamic acid as the matrix. The selected peaks were subjected to MS/MS analysis in LIFT mode.

To measure HYAL1 activity by in-gel digestion assay, we carried out the experiment as previously reported (26,27). Equal numbers (2.0×10⁶ cells) of HT1080-neo and HT1080-HYAL1-MH cells were cultured in serum-free DMEM for 24 h and concentrated by using Ni-NTA agarose. Ni-NTA-bound proteins were eluted, and the samples were electrophoresed on an SDS-polyacrylamide gel containing rooster comb HA (0.2 mg/ml; Sigma) at 4°C. The gel was washed twice with SDS extraction buffer (50 mM Tris-HCl, pH 7.5, 0.1 M NaCl, 2.5% Triton X-100) for 1 h and incubated in assay buffer (50 mM sodium formate, pH 4.0, 150 mM NaCl) at 37°C for 24 h. After incubation, the gel was stained with 0.5% alcian blue (Sigma) containing 20% ethanol and 10% acetic acid solution for 2 h and destained with 25% methanol and 7.5% acetic acid solution. HYAL1 activity can be observed as a transparent band in the blue pigment background.

We examined the conformational changes of HYAL1 when it was C-mannosylated and unmannosylated by using the Molecular Operating Environment (MOE; Chemical Computing Group Inc., Montreal, Canada) per previously reported methods (28,29). The Protein Data Bank sequences Apis melliflora HYAL-HA complex (PDB ID: 1FCV) and human HYAL1 (PDB ID: 2PE4) were loaded into MOE. Hydrogen atoms were added, and the protonation states were assigned using the Protonate 3D tool of MOE. Using AMBER12:EHT, one of the calculation methods of force field, energy was minimized. The 2PE4 sequence was aligned with 1FCV, removing the Apis melliflora HYAL and HA structure. Energy minimization was applied to human HYAL1 and HA. A 3D structure of the refined model was used for the Site Finder module of MOE, which can identify possible ligand-binding sites. Protein docking was performed by alpha sphere and excluded volume-based ligand-receptor docking (ASE-Dock), which is based on ligand-receptor interaction energies, and the resulting score was calculated as U_DOCK. U_DOCK is expressed by the sum of U_refine, the entire free energy of ligand-receptor interaction, and U_strain, the free energy of ligand stability. In the ASE-Dock module, ligand atoms have alpha spheres within 1 Å. Utilizing this property, models are created, and ligand atoms from many conformations that are generated by superposition with these points can be evaluated and scored by maximum overlap with alpha spheres and minimum overlap with the receptor atoms. Protein docking between human HYAL1 and dummy ligand atoms, which were created at chosen LBSs as centroids, was performed. The resulting U_DOCK was calculated. An α-D-mannose was properly added to Trp¹³⁰ of human HYAL1 to be C-mannosylated within MOE, and then the energy of C-mannosylated human HYAL1 was minimized. Protein docking between C-mannosylated human HYAL1 and dummy ligand atoms was performed. The resulting U_DOCK was calculated.

Human HYAL1 contains two predicted C-mannosylation consensus sequences, Trp¹³⁰ and Trp³²¹. The active site of HYAL1 (Glu¹³¹) is located next to Trp¹³⁰ (Fig. 1A). To examine whether HYAL1 is C-mannosylated or not, we established HT1080 cells that stably expressed HYAL1 (Fig. 1B).

HYAL1 has a signal peptide at the N-terminus domain (Fig. 1A) and is predicted to secrete. We examined whether HYAL1 secretes or not and confirmed HYAL1 secretion (Fig. 2A). Furthermore, secreted HYAL1 possessed enzymatic activity (Fig. 2A; see alcian blue staining). We undertook MALDI-TOF MS analysis to determine whether secreted HYAL1 is actually C-mannosylated (30). To obtain recombinant HYAL1 protein, we purified HYAL1 from conditioned medium of HT1080-HYAL1-MH cells by using Ni-NTA agarose (data not shown). Purified HYAL1 was treated with trypsin, and the resulting mixture of the peptides was analyzed by MALDI-TOF MS (Fig. 2B and C). C-mannosylation, the attachment of one mannose to a tryptophan residue, should provoke an increase of m/z 162. The peptides containing Trp¹³⁰ were observed at m/z 3,156.0, but no peaks around at m/z 3,318, which is the mass resulting from C-mannosylation of the peptide, were detected (Fig. 2B). In the same way, we performed MS analysis of the peptides containing Trp³²¹, and the peak was observed at m/z 6,181.3, which indicated that it was an unmannosylated peptide (Fig. 2C). However, the peak that was located at about m/z 6,343, which is the mass resulting from C-mannosylation of the peptide, was not detected (Fig. 2C). These results revealed that secreted HYAL1 was not C-mannosylated, although secreted HYAL1 possessed enzymatic activity.

Since HYAL1 is reported to lie mainly in lysosomes (31), we tried to examine whether intracellular HYAL1 is C-mannosylated. We purified HYAL1 proteins from whole-cell lysates of HT1080-HYAL1-MH cells (data not shown), and the obtained recombinant HYAL1 was treated with trypsin. The resulting mixture of peptides that were digested by trypsin was analyzed by MALDI-TOF MS (Fig. 3A and B). The peptides containing Trp¹³⁰ were observed at m/z 3,155.6, which were unmannosylated peptides (Fig. 3A). Moreover, a peak at m/z 3,317.7, which was an increase of m/z 162, was also observed (Fig. 3A). It suggested that the peptides containing Trp¹³⁰ were C-mannosylated. We performed MS analysis on the peptides containing Trp³²¹, and a peak was observed at only m/z 6,181.8, which was predicted to be an unmannosylated peptide (Fig. 3B). These results indicated that intracellular HYAL1 was C-mannosylated at only Trp¹³⁰. We further analyzed the peptides containing Trp¹³⁰ by MALDI-TOF MS/MS to confirm the mannose attachment residue (Fig. 3C and D). Unmannosylated peptides (Fig. 3C) and mannosylated peptides (Fig. 3D) were analyzed, and b4 and y5 ions were observed at the same position. However, y7 and y15 ions were observed at the m/z 162-increased positions in mannosylated peptides (Fig. 3D) compared with unmannosylated peptides (Fig. 3C). These results confirmed that C-mannosylation of HYAL1 occurred at Trp¹³⁰ only in the cell lysate, not the secreted HYAL1, suggesting that C-mannosylation might suppress HYAL1 secretion.

Since secreted HYAL1 showed enzymatic activity (Fig. 2A), C-mannosylation may not be essential for HYAL1 activity. However, since C-mannosylated Trp¹³⁰ lies next to Glu¹³¹, the active site of HYAL1, we assumed that C-mannosylation might regulate HYAL1 enzymatic activity. In order to examine the effect of C-mannosylation on HYAL1 enzymatic activity, we employed MOE, a computer simulation software that could calculate structural changes or free energies of interacting proteins. The crystal structure of human hyaluronidase-HA has not been reported, so we used the Protein Data Bank sequences for Apis melliflora hyaluronidase-HA complex (PDB: 1FCV) and human HYAL1 (PDB: 2PE4) in reference to a previous report (27) and performed protein alignment by the MOE tool to construct the human HYAL1-HA complex. The position of the active pocket of the constructed model accorded with 1FCV. We performed ASE-Dock between human HYAL1 and dummy atoms in the possible active pocket. As expected, HA was stably located at the active pocket, and the active site Glu¹³¹ nearly faced the HA cleavage site (Fig. 4A and B; docking energy, −38.6 kcal/mol). We next calculated the effect of C-mannosylation of HYAL1 on conformation or free energy. The docking simulation revealed that C-mannosylation changed the conformation, especially the nearby active pocket of HYAL1 (Fig. 4A and C). Moreover, the active site Glu¹³¹ faced in the opposite direction toward its substrate, HA (Fig. 4A and C; docking energy, −2.45 kcal/mol). These results suggest that C-mannosylation may cause the instability of HYAL1 conformation, resulting in suppression of HYAL1 enzymatic activity.