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

Abstract

Sperm lysozyme-like proteins belonging to c-type lysozyme family evolved in multiple forms. Lysozyme-like proteins, viz., 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 Lyzl 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.

Searching sequence databases for functional homologs using profile HMMs: how to set bit score thresholds?

“…Lysozyme-C family is named after hen egg white lysozyme, its prototypical member. This family includes lysozymes, lysozyme-like proteins and alpha-lactalbumins (26–29). Some lysozymes bind calcium [Ca(+)-Lys] whereas others do not [Ca(-)-Lys].…”

“…alpha-Lactalbumins have lost catalytic activity but have acquired a new function wherein, as part of the lactose synthase complex, they modify the acceptor substrate specificity of beta-1,4-galactosyltransferase (30). Phylogenetic analyses have indicated that alpha-lactalbumins and lysozymes diverged from a common ancestor, a calcium binding protein (28, 29) (Figure S1). The vestiges of divergence are evident in lysozymes that retain calcium-binding ability.…”

“…Lysozyme-C family includes lysozymes, lysozyme-like proteins and alpha-lactalbumins (Prager and Wilson 1988; Grobler, Rao, et al 1994; Irwin et al 2011; Kalra et al 2016). Some lysozymes bind calcium whereas others do not, denoted henceforth as Ca(+)-Lys and Ca(-)-Lys, respectively.…”

A mouse seminal vesicle‐secreted lysozyme c‐like protein modulates sperm capacitation

“…Most of the c‐type LYZs are primarily expressed in the testis 13,14,27–30 . Among them, LYZL4, 14 LYZL6, 13 and SPACA3, also named SLLP1, 12 are found to be acrosomal proteins and are implicated in sperm–oocyte fertilization.…”

Impact of the Microbiome on the Immune System

“…Tears help physically remove material from the eyes and contain lysozymes-hydrolytic enzymes that cleave glyosidic bonds in Nacetylmuramic acid and N-acetylglucosamine-which break down peptidoglycan in the cell walls of bacteria. 7 If a foreign molecule is able to make it past these initial barriers, then the immune system must respond accordingly.…”