Structural, Functional and Phylogenetic Analysis of Sperm Lysozyme-Like Proteins

<div><p>Sperm lysozyme-like proteins belonging to c-type lysozyme family evolved in multiple forms. Lysozyme-like proteins, <i>viz</i>., LYZL2, LYZL3 or SLLP1, LYZL4, LYZL5 and LYZL6 are expressed in the testis of mammals. Not all members of LYZL family have been uniformly and unambiguously identified in the genome and proteome of mammals. Some studies suggested a role of SLLP1 and LYZL4 in fertilization; however, the function of other LYZL proteins is unknown. We identified all known forms of LYZL proteins in buffalo sperm by LC-MS/MS. Cloning and sequence analysis of the <i>Lyzl</i> cDNA showed 38–50% identity at amino acid level among the buffalo LYZL paralogs, complete conservation of eight cysteines and other signature sequences of c-type lysozyme family. Catalytic residues in SLLP1, LYZL4 and LYZL5 have undergone replacement. The substrate binding residues showed significant variation in LYZL proteins. Residues at sites 62, 101, 114 in LYZL4; 101 in SLLP1; 37, 62, and 101 in LYZL6 were more variable among diverse species. Sites 63 and 108 occupied by tryptophan were least tolerant to variation. Site 37 also showed lower tolerance to substitution in SLLP1, LYZL4 and LYZL5, but more variable in non-testicular lysozymes. Models of LYZL proteins were created by homology modeling and the substrate binding pockets were analyzed in term of binding energies and contacting residues of LYZL proteins with tri-N-acetylglucosamine (NAG)₃ in the A-B-C and B-C-D binding mode. Except LYZL6, LYZL proteins did not show significant difference in binding energies in comparison to hen egg white lysozyme in the A-B-C mode. (NAG)₃ binding energy in the B-C-D mode was higher by 1.3–2.2 kcal/mol than in A-B-C mode. Structural analysis indicated that (NAG)₃ was involved in making more extensive interactions including hydrogen bonding with LYZL proteins in B-C-D mode than in A-B-C mode. Despite large sequence divergence among themselves and with respect to c-type lysozymes, substrate binding residues as well as hydrogen bonding network between (NAG)₃ and proteins were mostly conserved. LYZL5 in buffalo and other mammalian species contained additional 10–12 amino acid sequence at c-terminal that matched with ankyrin repeat domain-containing protein 27. Phylogenetic analysis indicated LYZL2 to be most ancient among all the LYZL proteins and that the evolution of LYZL proteins occurred through several gene duplications preceding the speciation of mammals from other vertebrates as distant as reptiles and amphibians.</p></div

Structural models of lysozyme-like proteins complexed with (NAG)₃ based on several template structures.

<p>PDB IDs of template are provided in material method part. For clarity only one model for each LYZL protein is shown. Panels a–LYZL2, b–SLLP1, c–LYZL4, d–LYZL5, e–LYZL6 and f– 1JEF (TEWL as one of the template). The protein part is shown in gray color and (NAG)₃ molecule in ball and stick style has been shown in elemental colors. The substrate binding residues interacting with (NAG)₃ are shown in three letter amino acid codes, while catalytic residues (residues at position 35 and 52 or 53) are labelled with single letter code. The NAG monomer binding subsites are represented by capital letters A, B and C in panel f.</p

Multiple sequence alignment of deduced amino acid sequences of buffalo matured LYZL2, SLLP1, LYZL4, LYZL5, LYZL6 and HEWL.

<p>The sequence shown within the red color box indicates specific signature of lysozyme family. The conserved cysteine and tryptophan residues are highlighted with yellow and green color bars, respectively. The catalytic residues corresponding to positions 35 and 52 of c-type lysozyme are shown in red color and underlined. The residues marked with diamond (♦) in blue color represent substrate binding sites in c-type lysozymes.</p

Binding energy, contacting residues and hydrogen bonding residues of LYZL with (NAG)₃ in their complex structures.

<p>Binding energy, contacting residues and hydrogen bonding residues of LYZL with (NAG)₃ in their complex structures.</p

Phylogenetic map of lysozyme family.

<p>The phylogenetic tree was constructed by using ML method. The nodes with diamond (♦) in blue color represent duplication event. LYZ-lysozyme, LYZL-lysozyme-like proteins, BoSt-cattle stomach, BuSt-buffalo stomach, ShSt-sheep stomach, BoMlk-cattle milk, BuMlk-buffalo milk, Hu-human, Ch-chimpanzee, Mc-monkey, Rt-rat, Mu-mouse, Bo-cattle, Bu-buffalo, Sh-sheep, Gt-goat, Py-python, Gr-garter snake. Lysozymes from non-mammals such as drosophila (Dr) and <i>Bombyx mori</i> (Bm) were used as outgroups.</p

Substrate binding residues in c-type lysozyme family.

<p>Substrate binding residues in c-type lysozyme family.</p

Hepatocyte growth on fibroblast feeder layer.

<p>Hepatocyte proliferation curve represented by the absorbance at 540 nm of BrdU-labelled hepatocytes at different days of culture.</p

Oil-red staining of lipid droplets in 5 days old cultured buffalo hepatocytes.

<p>Panel A shows lipid droplets (indicated by arrow) in hepatocytes in phase contrast at 200X magnification; and panel B shows oil red stained hepatocytes containing lipid droplets (arrow).</p

Temporal expression and secretion of albumin in condition media (CM) of hepatocytes.

<p>Panel A—Western blot analysis of albumin using condition media obtained from different time points of hepatocytes culture using FBS-free William’s E media. Lane 1: pre-stained protein marker; Lane 2: fresh hepatocytes lysate; Lane 3: 1^st day CM; Lane 4: 3^rd day CM; Lane 5: 4^th day CM; Lane 6: 5^th day CM; Lane 7: 7^th day CM. Panel B—Control experiment. Lane 1: pre-stained protein marker; Lane 2: pure hepatocytes (positive control); Lane 3: condition media (test); Lane 4: FBS-free William’s E media (negative control).</p

Immunostaining of 5 days old cultured buffalo hepatocytes with anti-cytokeratin-18 and anti-albumin.

<p>Immunostaining with (A) CY3 labelled anti-cytokeratin-18 antibodies (fluorescence signal in red); (B) FITC labelled anti-albumin antibodies (green); (C) staining of hepatocytes nuclei with DAPI (blue). Panel D shows the merged images from panels A, B and C. Panel E shows light microscopic image of hepatocytes; panel F shows negative control (Isotype control), and panels G shows staining with DAPI, while panel H shows images merged from panels F and G.</p