BOR - Papers in Press, published online ahead of print
July 13, 2005.
Biol Reprod 2005, 10.1095/biolreprod.105.041889
BIOLOGY OF REPRODUCTION 73, 1064–1071 (2005)
DOI: 10.1095/biolreprod.105.041889
© 2005 by the Society for the Study of Reproduction, Inc.
Molecular Cloning and Characterization of Three Novel Lysozyme-Like Genes, Predominantly Expressed in the Male Reproductive System of Humans, Belonging to the C-Type Lysozyme/Alpha-Lactalbumin Family1
Kexiong Zhang ,
Rui Gao ,
Haoxing Zhang ,
Xin Cai ,
Chunhua Shen ,
Chaoqun Wu ,
Shouyuan Zhao , and
Long Yu 2
State Key Laboratory of Genetic Engineering, Institute of Genetics, School of Life Sciences, Fudan University, Shanghai 200433, People's Republic of China
 |
ABSTRACT
|
|---|
Lysozymes, especially c-type lysozymes, are well-recognized bacteriolytic factors widely distributed in the animal kingdom and play a mainly protective role in host defense. The relatives of c-type lysozymes, alpha-lactalbumins, however, are only found in mammalian milk and possess a distinct biological function. These two proteins, having similar amino acid sequences, gene structure, and dimensional conformation, belong to the c-type lysozyme/alpha-lactalbumin family. Using human lysozyme as an information probe, we cloned four human cDNAs encoding homologues of human lysozyme; these were named LYZL2, LYZL4, LYZL6, and SPACA3 by the HUGO Gene Nomenclature Committee. Of these four, SPACA3 has been reported to code an intra-acrosomal sperm protein SLLP1. To our knowledge, the other three are reported here for the first time. Using Northern blot hybridization, including 16 different human tissues, we found that these four lysozyme-like genes were all highly expressed in the testis/epididymis. Further analysis of one, LYZL4, by in situ hybridization revealed that its mRNA was only detected in the epithelium of human epididymis, most abundantly in the caput, suggesting that LYZL4 plays a physiological role in male reproduction. By sequence analysis, we found that two essential catalytic residues of the human lysozyme were conserved in LYZL2 and LYZL6, whereas one site in LYZL4 and two sites in SPACA3 were replaced. The LYZL2, LYZL4, LYZL6, and SPACA3 genes were mapped to human chromosome 10p11.23, 3p21.33, 17q11.2, and 17q12, respectively, and displayed a similar genomic structure. Our data suggest that these four lysozyme-like genes, which have arisen from a common progenitor gene, play a major role in human reproduction.
epididymis, gene regulation, male reproductive tract, male sexual function, testis
|
INTRODUCTION
|
|---|
More than 70 yr ago, Alexander Fleming discovered a remarkable bacteriolytic element, for which he coined the name lysozyme [1]. Intensive investigations have led to the identification of many lysozymes in nature, and these have been classified into several categories based mainly on their molecular characteristics [2, 3]. Of them, the c-type (chicken type) lysozymes have been well studied and shown to have a wide distribution, including mammals, birds, reptiles, fishes, and insects [2]. In fact, the c-type lysozymes are enzymes that catalyze the hydrolysis of ß-1,4 glycosidic bonds of the peptidoglycan of bacterial cell walls, and this accounts for their main biological function of protecting the host from bacterial infection [2]. Recent evidence supports the catalysis mechanism of c-type lysozymes as involving a covalent intermediate in which Glu-35 and Asp-52, the two active residues, interact with the ß-1,4 glycosidic bond of the substrate [4]. The alpha-lactalbumins (known throughout as 
-lactalbumins), however, are found only in mammalian milk and have a function distinct from that of the c-type lysozymes; that is, they act as a regulatory component by modulating the carbohydrate-binding properties of the ß-1,4-galactosyltransferase through a specific protein-protein interaction so that together, they can catalyze the biosynthesis of lactose in the lactating mammary gland [5]. Although displaying a distinguishing biological function, the -lactalbumins have a close relationship with the c-type lysozymes in their amino acid sequence, gene structure, and dimensional conformation, resulting in their placement in the c-type lysozyme/-lactalbumin family. Furthermore, accumulating evidence suggests that their coded genes have evolved from a common progenitor [6–8].
As a c-type lysozyme, human lysozyme has been studied extensively. Being widely expressed in human kidney, stomach, small intestine, lacrimal gland, parotid gland, sublingual gland, lung, spleen, lymph node, placenta, and leukocytes, human lysozyme exhibits various biological activities, including bactericidal defense, chemotaxis, protection against infection with human immunodeficiency virus, and association with tumors [9–16]. However, two earlier systematic investigations in humans using immunohistochemistry methods suggested that human lysozyme was not expressed in the testicular tissue [9, 10].
In the mammalian male reproductive system, sperm are produced in the seminiferous tubules of the testis and then transported to the epididymis, a long and convoluted tubule that is divided into three major regions: caput, corpus, and cauda. During their journey through the epididymis, spermatozoa are exposed to a microenvironment created by the absorptive and secretory activities of the epididymal epithelium [17]. The main physiological role of the epididymis involves sperm maturation as well as sperm protection and storage [18–20].
A human lysozyme-like protein, SLLP1, was identified recently as an intra-acrosomal sperm protein, and its encoded gene, SPACA3, was cloned [21]. In the present study, using human lysozyme as an information probe, we isolated four lysozyme-like cDNAs from the human testis cDNA library. One of these was demonstrated to be SPACA3, and the other three were novel genes. By Northern blot analysis, we found that all four are predominantly expressed in human testis/epididymis. Moreover, further investigation by in situ hybridization revealed that LYZL4 was specifically highly expressed in the epithelium of human caput epididymis. The similarities in the amino acid sequence and genomic structure of these four lysozyme-like genes suggest that they arose from a common progenitor.
|
MATERIALS AND METHODS
|
|---|
cDNA Cloning and Sequencing
Using the nucleotide sequence of human lysozyme as a query for a BLAST search at the human expressed sequence tag (EST) database of GenBank, we obtained homologous ESTs, assembled them as contigs, and identified open reading frames (ORFs). Based on the contig sequence, we designed primers and used them to amplify cDNAs from a human testis library. After labeling polymerase chain reaction (PCR) fragments with [32P]deoxycytidine triphosphate by a random primer method, a human testis 
cDNA library (Clontech) was screened twice for positive phage plaques. Using purified positive plaques as templates with gene-specific and vector-specific primers, the cDNA inserts were amplified by PCR and cloned into the pGEM-T vector (Promega) for sequencing. Integration of the sequencing data yielded an approximately full-length sequence of each cDNA obtained by screening the cDNA library.
Northern Blot Analysis
Human multiple-tissue Northern membranes (MTN I and MTN II) containing mRNAs from 16 tissues were purchased from Clontech and used to perform hybridization with the LYZL2, LYZL4, LYZL6, and SPACA3 probes, under conditions described previously [22]. The probes were prepared by labeling the cDNA fragments (amplified from human cDNA libraries as mentioned above) with [-32P]dATP (Amersham Biosciences), using PCR and purified by a Sepharose G50 column. The same MTN I or MTN II membranes were hybridized with human ß-actin cDNA to demonstrate equal loading and exposed to radiographic film (Eastman Kodak) at –80°C for 8 h.
Tissue Preparation
Human testis tissue for in situ hybridization was obtained through our local organ transplantation program. The testis tissue was obtained from a donor immediately after his death. This donor was without medical antecedents affecting reproductive function. The sampling and procedures were preapproved by our local ethics committee. The testis tissue was fixed quickly after removal by immersion in Bouin fixative for 4–12 h, depending on the tissue size. The epididymis was excised and dissected into caput, corpus, and caudal regions. After fixation, tissues were dehydrated in a series of increasing concentrations of ethanol and then processed for paraffin embedding and sectioning. Thin sections (thickness, 5 µm) were cut, mounted onto poly-L-glycine-coated slides, and dried. All procedures were conducted under RNase-free conditions.
In Situ Hybridization
An in situ hybridization procedure was performed using digoxigenin (DIG)-labeled RNA probes as described previously [23]. The lengths of antisense and sense probes were identical, with both corresponding to the same region consisting of part 3'-terminal ORF and part 3'-untranslated region sequence of LYZL4.
First, slides were deparaffinized by two changes of xylene, followed by rehydration in a series of decreasing concentrations of ethanol, followed by successive treatment with Bouin fixative for 5 min. After digestion and acetylation, the sections were then prehybridized for 2 h at 50°C, with a hybridization buffer (40% deionized formamide, 1x Denhardt solution, 4x SSC [single strength: 0.15 M sodium chloride and 0.015 M sodium citrate], 10 mM dithiothreitol, 1 mg/ml of yeast tRNA, and 1 mg/ml of denatured and sheared salmon sperm DNA). Hybridization was carried out for 12–16 h at 50°C in the above-mentioned hybridization buffer with further addition of 10% dextran sulfate and 2 mg/ml of heat-denatured DIG-labeled cRNA probe. The washing procedure for RNase-A digestion has been described previously [24]. For immunological detection with the DIG Nucleic Acid Detection Kit, the slides were washed twice with PBS (10 min for each wash), blocked with blocking solution (containing 0.1% Triton X-100 and 2% normal sheep serum) for 30 min, and then incubated with buffer containing 0.1% Triton X-100, 1% normal sheep serum, and alkaline phosphatase-labeled anti-DIG antibody (1:1000) for 2 h. Thereafter, the slides were first washed four times with PBS (5 min for each wash), twice with buffer 1 (10 mM Tris-HCl [pH 8.0], 50 mM NaCl, and 10 mM MgCl2; 5 min for each wash), and finally twice with buffer 2 (10 mM Tris-HCl [pH 9.5] and 10 mM MgCl2; 5 min for each wash). Thereafter, the hybridization signal was visualized with buffer 2 containing nitroblue tetrazolium-5-bromo-4-chloro-3-indolylphosphate (1:50) and 1 mM levamisole. The color reaction was stopped by washing the slides with buffer 3 (10 mM Tris-HCl [pH 7.4] and 1 mM EDTA). Hematoxylin-and-eosin (HE) stains were done to serve as histological controls for in situ hybridization.
Sequences Analysis
We performed a homology search using the BLAST algorithms against the GenBank database (http://www.ncbi.nlm.nih.gov/BLAST/). Signal peptides were predicted by SignalP (http://www.cbs.dtu.dk/services/SignalP/). The multiple sequence alignments were performed by ClustalW (http://www.ebi.ac.uk/clustalw/) and BOXSHADE (http://www.ch.embnet.org/software/Box_form.html) programs in default settings. The phylogenetic tree was carried out with the ClustalW program at the website (http://clustalw.genome.jp/).
|
RESULTS
|
|---|
cDNA Cloning, Sequence Analysis, and Genomic Organization of Four Human Lysozyme-like Genes: LYZL2, LYZL4, LYZL6, and SPACA3
By BLAST-searching the human EST database of GenBank, using the nucleotide sequence of human lysozyme as a query, we obtained a series of homologous ESTs and assembled them as four contigs. By screening a human testis cDNA library, sequencing the positive clones and integrating these data, four cDNA sequences, each containing a complete ORF, were obtained and deposited in GenBank as accession numbers AF326749, AF099029, AF139543, and AY742214, and named LYZL4 (also known as LYC4), SPACA3 (also known as LYC3 and LYZL3), LYZL2, and LYZL6 (also known as LYC1), respectively, by the HUGO Gene Nomenclature Committee. All four proteins are predicted to contain N-terminal signal peptides of 18–19 amino acids [25, 26] (Figs. 1 and 2).
View larger version (78K):
[in this window]
[in a new window]
|
FIG. 1. Nucleotide sequences, predicted ORFs of lysozyme-like SPACA3, and three novel lysozyme-like cDNAs. The translation of the proposed ORF is shown below the nucleotide sequence. The amino acid numbers are shown on the left, and the signal sequences are shaded. The positions of the translational stop codons are marked by asterisks. Polyadenylation signals ( ) are underlined
|
|
By BLAST-searching the GenBank database of human genes and proteins, the most homologous sequence to these four proteins was found to be human lysozyme, and the second was human -lactalbumin. As shown in Figure 2 and Table 1, the four proteins are considerably homologous to each other. The alignment (Fig. 2) revealed that one of the unique features of these four proteins is the presence of eight cysteine residues that are conserved in all members of the c-type lysozyme/-lactalbumin family. The two essential residues at positions 35 and 52 in the c-type lysozymes were conserved in human LYZL2 and LYZL6, whereas one amino acid of LYZL4 and two amino acids of SPACA3 were replaced (Fig. 2 and Table 2).
View this table:
[in this window]
[in a new window]
|
TABLE 2. Comparison of the characteristics of LYZL2, LYZL4, LYZL6 and SPACA3 with some c-type lysozymes and -lactalbumins
|
|
To better understand how these four proteins are related to other c-type lysozymes and -lactalbumin, a phylogenetic tree was constructed (Fig. 3). The tree suggested a probable duplication of an ancestral gene to form the -lactalbumin lineage, which includes equine milk calcium-binding lysozyme, and a second lineage that further duplicated and diverged to form two distinct clades, one including the LYZL (LYZL2, LYZL4, LYZL6, and SPACA3) and the other c-type lysozymes.
View larger version (16K):
[in this window]
[in a new window]
|
FIG. 3. The phylogenetic tree. This polygenetic tree was constructed by the ClustalW software using the bootstrap method. B-LALBA, bovine -lactalbumin, P00711; Ce-LYZ, chicken egg white lysozyme, LZCH; Em-LYZ, equine milk calcium-binding lysozyme, P11376; H-LALBA, human -lactalbumin, P00709; H-LYZ, human lysozyme, P00659; Ls-HLZ, langur monkey stomach lysozyme, P67977; Ss-LYZ, sheep stomach lysozyme, P17607; Tr-LYZ, trout lysozyme, P11941
|
|
To determine the genomic organization, the LYZL2, LYZL4, LYZL6, and SPACA3 genes were mapped by BLAST-searching against the human genome database at NCBI to chromosome 10p11.23, 3p21.33, 17q11.2, and 17q12, respectively. The genomic structures of these four genes, including the sequences immediately flanking the exon-intron junctions, are shown in Figure 4. All the introns conformed to the GT/AG rule. When viewed in vertical alignment, to compare the genomic structure of these four human genes and human lysozyme gene LYZ, we found that the length of the corresponding coding parts were almost identical and that the positions interrupted by introns and the amino acid spanning the spliced introns were very similar (Fig. 4).
View larger version (24K):
[in this window]
[in a new window]
|
FIG. 4. The genomic structures of four human lysozyme-like genes and human lysozyme gene. The positions interrupted by intron splicing are shown. Exons (boxes) are drawn to scale. Shadows denote the coding parts, whereas gray shadows represent the signal peptides
|
|
LYZ2, LYZL4, LYZL6, and SPACA3 Genes Are Predominantly Expressed in the Testis/Epididymis by Northern Blot Analysis
To determine the overall expression profile of these four human genes, Northern blot hybridizations using membranes containing mRNA from 16 different human tissues were performed, using the labeled human cDNA fragments of LYZL2, LYZL4, LYZL6, and SPACA3 as hybridizing probes. Interestingly, all mRNAs of these four genes were highly expressed in testis/epididymis as an approximately 1.0-kilobase transcript in the tested tissues, which was in good agreement with the size of each cloned cDNA plus the addition of the poly(A)+ tail. Representative results are illustrated in Figure 5. Both LYZL6 and LYZL4 mRNAs were specifically expressed in testis/epididymis, and no detectable signals were found in other tissues. In addition to a strong signal of LYZL2 expression in testis/epididymis, a weak band with the same size also was observed in placenta. The expression of SPACA3 was similar to that of LYZL2; the mRNA was expressed strongly in testis/epididymis and weakly in pancreas. In line with these results, a search against the EST database using these four gene sequences revealed many matches to ESTs of human testis/ epididymis origin for each.
View larger version (72K):
[in this window]
[in a new window]
|
FIG. 5. Northern blot analyses of the four human lysozyme-like genes. With each gene, the membrane preloaded with mRNA from 16 human tissues was purchased from Clontech and hybridized with a 32P-labeled probe. ß-Actin was used as a control
|
|
LYZL4 mRNA Is Exclusively Expressed in the Epithelium of Human Epididymis
Northern blot analyses were performed to examine the distribution of LYZL4 mRNA in different human tissues. The LYZL4 mRNA was only detected in the testis as an approximately 1.0-kilobase, single transcript, whereas it was absent in the other 15 tissues, including heart, brain, placenta, lung, liver, skeletal muscle, kidney, pancreas, spleen, thymus, prostate, ovary, small intestine, colon, and peripheral blood leukocyte on a multiple-tissue Northern blot. To further investigate the expression pattern of human LYZL4 mRNA, in situ hybridization was performed in testis sections and the different parts of the human epididymis. As shown in Figure 6, the expression of LYZL4 mRNA was strong in the epididymis, whereas no obvious expression was detected in the seminiferous tubules of the testis. Thus, the human testis RNA used in the commercial Northern blot may have contained RNA from the epididymis. In the human epididymis, strong expression of LYZL4 mRNA was found in the epithelium of the caput, and weak expression was detected in the corpus and caudal epithelia (Fig. 6). Epithelial location was confirmed by HE staining.
View larger version (101K):
[in this window]
[in a new window]
|
FIG. 6. With in situ hybridization analysis, a strong expression of LYZL4 was observed in human caput epididymis. Paraffin sections from the seminiferous tubules of testis (A), proximal caput (B), distal caput (C), proximal corpus (J), distal corpus (K), and caudal epididymis (L) were hybridized with LYZL4 DIG-labeled antisense probe. Control hybridization experiments (D–F and M–O) were performed using a sense LYZL4 RNA probe in a similar region corresponding to antisense hybridization. The HE stains were done in corresponding regions (G–I, and P–R). Magnification, x20
|
|
|
DISCUSSION
|
|---|
It has been known for many years that humans have one lysozyme that is expressed ubiquitously in the human body. Recently, by two-dimensional electrophoresis and further proteomics analysis, a lysozyme-like human SLLP1 was identified as a male reproduction-associated protein, and its encoded cDNA was cloned and named SPACA3 [21]. In the present study, using human lysozyme as an information probe, we cloned four lysozyme-like cDNAs: One is SPACA3, and the others are three novel human cDNAs (LYZL2, LYZL4, and LYZL6). This indicates that more than one lysozyme-like molecule is expressed in the human body. Although they display considerable sequence divergence compared to human lysozyme or -lactalbumins, the signature of the eight conserved cysteine residues supports the classification of these lysozyme-like proteins into the c-type lysozyme/-lactalbumin family. In fact, based on homology difference analysis, these four lysozyme-like proteins are closer to lysozymes than to -lactalbumin.
With the help of bioinformatics analysis, we successfully cloned the corresponding orthologs of these four genes from a mouse cDNA library (data not shown). Using BLAST-searching against the rat genomic database, a significant homology in the sequences also was found. However, in a released database of other species, including chicken, drosophila, Caenorhabditis elegans, yeast, and Escherichia coli, we did not detect obvious homologous sequences, suggesting that these four molecules (LYZL2, LYZL4, LYZL6, and SPACA3) may exert their physiological functions in mammals.
Unlike the human lysozyme and mammalian -lactalbumins, these four human lysozyme-like genes show a different expression pattern; that is, they are highly expressed in human testis/epididymis by Northern blot analysis, suggesting the association of these molecules with human reproduction. Of these, LYZL4 is specifically expressed in the human testis/epididymis. In our in situ hybridization experiments, we did not detect the expression of LYZL4 in the seminiferous tubules of the testis, but a strong signal was observed in the epithelium of the caput epididymis. This observation, taken together with the predicted signal peptide of LYZL4, makes it likely that LYZL4 is a protein secreted by the epithelium of the caput epididymis and may function in the epididymis. The proximal segment region of the epididymis is particularly important [27–29] in that the secretory proteins are likely to initiate events within the luminal fluid or spermatozoa that ultimately are expressed (physiologically) as sperm motility or fertility in more distal epididymal regions. In contrast to other regions, the proximal segment is acutely sensitive to undefined factors from the testis, the absence of which results in downregulation of a subset of genes with putative roles in early sperm maturation [27, 28]. Furthermore, the caput epididymis is a region in which several antimicrobial proteins have been identified, such as Binb1 [30], ESC42 (or DEFB118) [31, 32], and HE2 [33–35]. The specific, high expression of LYZL4 in human caput epididymis suggests an important role for LYZL4 in male reproduction.
The similar characteristics of LYZL2, LYZL4, LYZL6, SPACA3, and human lysozyme in the composition of amino acid, genomic structure, and signal peptide suggest that these four lysozyme-like genes have a common progenitor with human lysozyme. In fact, the lysozymes are considered to be ancient protective proteins and are widely distributed in nature, from prokaryotes to insects, reptiles, and mammals. Based on our bioinformatics search, LYZL2, LYZL4, LYZL6, SPACA3, and their orthologs are found only in mammals, suggesting that these lysozyme-like genes appeared at a relatively later time as a result of gene duplication of the lysozyme. Similarly, molecular evolution analysis has provided evidence to support the suggestion that gene duplication of c-type lysozyme gave rise to -lactalbumin before birds and mammals diverged some 300 million years ago [7, 8]. It may be reasonable to conclude that the first gene duplication giving rise to these four lysozyme-like genes occurred after that time, considering that these four lysozyme-like proteins are more homologous to mammalian c-type lysozymes than to -lactalbumins.
A review of the literature revealed that SLLP1, which recently was described as an intra-acrosomal sperm protein [21], is encoded by the SPACA3 gene. Two essential residues of human lysozyme were replaced in SLLP1(SPACA3), resulting in a loss of the bacteriolytic activity and the function involved in sperm-egg binding [21]. However, based on a comparison of the amino acid sequences, we found that the two essential catalytic residues, 35-Glu and 52-Asp of human lysozyme, or all the c-type lysozymes, including the conventional and calcium-binding lysozymes, were conserved in both LYZL2 and LYZL6, suggesting that these two proteins may retain the catalytic bacteriolytic activity and play a role in host defense against infection in the male reproductive system of humans. LYZL4, however, with one essential residue (35-Glu) retained and another residue (52-Asp) replaced, is different from either LYZL2 and LYZL6 or SLLP1 and -lactalbumins, making its biological activities elusive. The two essential catalytic residues are both "mutated" in SLLP1 and -lactalbumins, which may account for the altered function of these proteins. It is plausible that SLLP1 is involved in sperm-egg binding and that -lactalbumins act as a regulating factor of lactose synthesis in the lactating mammary gland rather than displaying bactericidal activity in host defense. Further investigation of the physiological roles in reproduction and the bacteriolytic activity of these newly identified, lysozyme-like molecules is urgently needed to elucidate the molecular evolution and related functional divergence of the c-type lysozyme/-lactalbumin family.
|
FOOTNOTES
|
|---|
1 Supported by National 973 Program (grant 973-010203) and National Natural Science Foundation of China (grant 30024001). GenBank accession numbers AF 326749, AF 099029, AF 139543, and AY 742214. 
2 Correspondence: Long Yu, State Key Laboratory of Genetic Engineering, Institute of Genetics, School of Life Sciences, Fudan University, Shanghai 200433, People's Republic of China. FAX: 86 21 65643250; longyu{at}fudan.edu.cn
Received: 13 March 2005.
First decision: 11 April 2005.
Accepted: 8 July 2005.
|
REFERENCES
|
|---|
-
Fleming A. On a remarkable bacteriolytic element found in tissues and secretions. Proc R Soc Lond B Biol Sci 1922 93:306-317[Free Full Text]
-
Jolles P, Jolles J. What's new in lysozyme research? Always a model system, today as yesterday. Mol Cell Biochem 1984 63:165-189[CrossRef][Medline]
-
Ito Y, Yoshikawa A, Hotani T, Fukuda S, Sugimura K, Imoto T. Amino acid sequences of lysozymes newly purified from invertebrates imply wide distribution of a novel class in the lysozyme family. Eur J Biochem 1999 259:456-461[Medline]
-
Vocadlo DJ, Davies GJ, Laine R, Withers SG. Catalysis by hen egg-white lysozyme proceeds via a covalent intermediate. Nature 2001 412:835-838[CrossRef][Medline]
-
Brew K, Hill RL. Lactose biosynthesis. Rev Physiol Biochem Pharmacol 1975 72:105-158[Medline]
-
McKenzie HA, White FH Jr. Lysozyme and -lactalbumin: structure, function, and interrelationships. Adv Protein Chem 1991 41:173-315[Medline]
-
Prager EM, Wilson AC. Ancient origin of lactalbumin from lysozyme: analysis of DNA and amino acid sequences. J Mol Evol 1988 27:326-335[CrossRef][Medline]
-
Nitta K, Sugai S. The evolution of lysozyme and -lactalbumin. Eur J Biochem 1989 182:111-118[Medline]
-
Klockars M, Reitamo S. Tissue distribution of lysozyme in man. J Histochem Cytochem 1975 23:932-940[Abstract]
-
Mason DY, Taylor CR. The distribution of muramidase (lysozyme) in human tissues. J Clin Pathol 1975 28:124-132[Abstract/Free Full Text]
-
Osserman EF, Canfield RE, Beychok S. Lysozyme. New York: Academic Press; 1974
-
Klockars M, Roberts P. Stimulation of phagocytosis by human lysozyme. Acta Haematol 1976 55:289-295[CrossRef][Medline]
-
Bodansky O. Blood enzymes in cancer and other diseases. Adv Cancer Res 1961 6:1-80[Medline]
-
Luger T, Kokoschka EM, Sagaster P, Micksche M. Serum lysozyme levels in patients with solid tumors. Oncology 1979 36:15-18[Medline]
-
Caruso RA, Spada FL, Casablanca G, Rigoli L. Lysozyme and mucins in gastric adenomas. J Clin Pathol 1989 42:827-833[Abstract/Free Full Text]
-
Lee-Huang S, Huang PL, Sun Y, Huang PL, Kung H, Blithe DL, Chen HC. Lysozyme and RNases as anti-HIV components in ß-core preparations of human chorionic gonadotropin. Proc Natl Acad Sci U S A 1999 96:2678-2681[Abstract/Free Full Text]
-
Cooper TG. Interactions between epididymal secretions and spermatozoa. J Reprod Fertil Suppl 1998 53:119-136[Medline]
-
Turner TT. On the epididymis and its role in the development of the fertile ejaculate. J Androl 1995 16:292-298[Free Full Text]
-
Hinton BT, Palladino MA, Rudolph D, Labus JC. The epididymis as protector of maturing spermatozoa. Reprod Fertil Dev 1995 7:731-745[CrossRef][Medline]
-
Moore HD. Contribution of epididymal factors to sperm maturation and storage. Andrologia 1998 30:233-239[Medline]
-
Mandal A, Klotz KL, Shetty J, Jayes FL, Wolkowicz MJ, Bolling LC, Coonrod SA, Black MB, Diekman AB, Haystead TAJ, Flickinger CJ, Herr JC. SLLP1, a unique, intra-acrosomal, nonbacteriolytic, c lysozyme-like protein of human spermatozoa. Biol Reprod 2003 68:1525-1537[Abstract/Free Full Text]
-
He H, Dang Y, Dai F, Guo Z, Wu J, She X, Pei Y, Chen Y, Ling W, Wu C, Zhao S, Liu JO, Yu L. Posttranslational modifications of three members of the human MAP1LC3 family and detection of a novel type of modification for MAP1LC3B. J Biol Chem 2003 278:29278-29287[Abstract/Free Full Text]
-
Zhang KX, Yu L, Sun QW, Zhu TF, Saiyin H, Zhou GJ, Wu CQ, Zhao SY. Expression of Cdv-1R gene in mouse epididymis as revealed by in situ hybridization. Arch Androl 2005 51:7-13[CrossRef][Medline]
-
Komminoth P. Digoxigenin as an alternative probe labeling for in situ hybridization. Diagn Mol Pathol 1992 1:142-150[Medline]
-
Bendtsen JD, Nielsen H, Heijne G von, Brunak S. Improved prediction of signal peptides: SignalP 3.0. J Mol Biol 2004 340:783-795[CrossRef][Medline]
-
Nielsen H, Engelbrecht J, Brunak S, Heijne G von. Identification of prokaryotic and eukaryotic signal peptides and prediction of their cleavage sites. Protein Eng 1997 10:1-6[Abstract/Free Full Text]
-
Cornwall GA, Hann SR. Specialized gene-expression in the epididymis. J Androl 1995 16:379-383[Free Full Text]
-
Hinton BT, Lan ZJ, Rudolph DB, Labus JC, Lye RJ. Testicular regulation of epididymal gene expression. J Reprod Fertil Suppl 1998 53:47-57[Medline]
-
Hsia N, Cornwall GA. DNA microarray analysis of region-specific gene expression in the mouse epididymis. Biol Reprod 2004 70:448-457[Abstract/Free Full Text]
-
Li P, Chan HC, He B, So SC, Chung YW, Shang Q, Zhang YD, Zhang YL. An antimicrobial peptide gene found in the male reproductive system of rats. Science 2001 291:1783-1785[Abstract/Free Full Text]
-
Yenugu S, Hamil KG, Radhakrishnan Y, French FS, Hall SH. The androgen-regulated epididymal sperm-binding protein, human ß-defensin 118 (DEFB118) (formerly ESC42), is an antimicrobial ß-defensin. Endocrinology 2004 145:3165-3173[Abstract/Free Full Text]
-
Liu Q, Hamil KG, Sivashanmugam P, Grossman G, Soundararajan R, Rao AJ, Richardson RT, Zhang YL, O'Rand MG, Petrusz P, French FS, Hall SH. Primate epididymis-specific proteins: characterization of ESC42, a novel protein containing a trefoil-like motif in monkey and human. Endocrinology 2001 142:4529-4539[Abstract/Free Full Text]
-
Yenugu S, Hamil KG, Birse CE, Ruben SM, French FS, Hall SH. Antibacterial properties of the sperm-binding proteins and peptides of human epididymis 2 (HE2) family; salt sensitivity, structural dependence, and their interaction with outer and cytoplasmic membranes of Escherichia coli. Biochem J 2003 372:473-483[CrossRef][Medline]
-
Horsten HH, Derr P, Kirchhoff C. Novel antimicrobial peptide of human epididymal duct origin. Biol Reprod 2002 67:804-813[Abstract/Free Full Text]
-
Osterhoff C, Kirchhoff C, Krull N, Ivell R. Molecular cloning and characterization of a novel human sperm antigen (HE2) specifically expressed in the proximal epididymis. Biol Reprod 1994 50:516-525[Abstract]
TOP
ABSTRACT
INTRODUCTION
