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| | Structure of human PSAP, saposins, and sequence alignment of a known neurotrophic fragment of human saposin C.
(A) Organization of human PSAP protein. Individual saposin domains and signal peptide are indicated; lightning bolts represent proteolytic cleavage sites in the intersaposin sequences; glycosylation sites and exon-intron boundaries are shown by 8-point stars and vertical lines, respectively.
(B) Amino acid sequence of human saposin A-D. Potential N-Glycosylation type carbohydrate side chain linked to asparagine are indicated with capital letter "N". As indicated, each saposin molecule also contains 6 cystein residues positioned at almost similar location. Neurotrophic sequence of saposin C is double-underlined.
(C) Alignment and comparison of neurotrophic sequence of human saposin C with other vertebrates and known viruses. All sequences presented are linear. Each plus sign indicates the presence of a non-fit amino acid. DESCRIPTION Prosaposin is a highly conserved glycoprotein (with approximate molecular weight of 65-72 kDa), and the precursor of 4 small lysosomal proteins (saposin A-D; of 8-13 kDa) which are required for intracellular degradation of certain sphingolipids. Proteolytic cleavage of PSAP precursor mediated by lysosomal cysteine protease-cathepsin D, leads to individual mature saposin proteins (acidic glycoproteins).PSAP is secreted as a full-length protein. However, individual saposin proteins also exist as extracellular mature proteins (e.g., in tissue culture supernatant, serum, prostatic secretions, malignant pleural effusion). Although the origin of mature saposin proteins in the extracellular fluids is not known, it is likely that circulating serum enzymes may participate in proteolytic cleavage of secreted PSAP. Each saposin domain presents with near identical localization of glycosylation sites and cysteine residues. The presence of high percentage homology in amino acid sequences between saposin A and C further indicates that they have originated from a single ancestroral gene at least via duplication and/or gene rearrangement. |
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| Description | Prosaposin is the saposin precursor protein with 524 amino acids including a 16 amino acids signal peptide. The full-length precursor molecule contains complex oligosaccharides chains which is probably the result of cotranslational glycosylation of the 53-kDa polypeptide and its later modification within the Golgi system that yield the 70-72 kDa precursor protein. After transport to the lysosome, cathepsin D participates in its proteolytic processing to intermediate molecular forms with 35 to 53 kDa and then to 13-kDa glycoprotein and finally to the mature 8-11 kDa less or partially glycosylated forms of individual saposin molecules. It is noteworthy that western analysis (using different anti-PSAP monoclonal and polyclonal antibodies) of human seminal fluid and whole cell lysates prepared from a number of malignant prostatic cells and other malignant cell types (e.g., breast, lung) show the presence of multiple bands with approximate molecular weight of 12-, 24-, and 36-kDa. These bands are most probably represent mono-, di-, and tri-saposins and are the result of sequential cleavage of the precursor molecule. Saposins are highly homologous molecules, each with approximately 80 amino acids containing six cysteine residues (forming 3 disulfide bonds and hair-pin structure) and N-glycosylated carbohydrate chains that are highly conserved. PSAP amino acid sequence among various species (e.g., human, rat, mouse, chicken, Zebrafish) reveals evolutionary conservation in terms of saposin domains and the homologus positioning of terminally-situated cysteine residues and an N-linked glycosylated site. |
| Expression | Prosaposin and individual saposin proteins are expressed by a wide variety of cells types originating from ectodermal, mesodermal, and endodermal germ layers including but not limited to lung, skin, fibroblast, stromal cells, bone, smooth muscle, skeletal muscle, cardiac muscle, placenta, red and white blood cells, pancreas, placenta, lymphoreticular system (spleen, thymus, liver), micro and macrovascular system, genitourinary system (e.g., prostate, testes, seminal vesicle), central and peripheral nervous system, etc. Interestingly, comparative protein expression analysis on normal human adult and fetal tissues has shown elevated levels of PSAP expression in the adult liver and decreased amounts in fetal skeletal muscle. Prosaposin and saposins also present as soluble proteins in extracellular space/fluid including pleural fluid, cerebrospinal fluid, seminal fluid, milk, and serum. PSAP and saposins are predominantly expressed in cells of hematopoietic origin (e.g., red- and white-blood cells) and neuroglial-derived tissues as compared to all other normal cell types in the mammalian system. In malignant cells, compared to their normal cellular counterparts, prosaposin is overexpressed in breast adenocarcinoma cell lines, non small-cell lung adenocarcinoma, neuroblastoma, and schwannoma cell lines. In addition, similar PSAP-overexpression is also detected in glioma cell lines, adult and pediatric brain tumors (e.g., medulloblastoma-, astrocytoma-, glioblastoma multiforme-cell lines), fibrosarcoma, osteosarcoma, and prostate cancer cell lines. In addition, immunoblotting of total protein array derived from different types of tumors (brain, colon, lung, pancreas, rectum, ovary, parotid, skin, bladder, small intestine, thymus, and uterus) with mouse monoclonal antibodies against PSAP and GAPDH followed by densitometric analysis demonstrated 1.6 to 5-fold increase in PSAP expression in malignant tissues compared to their corresponding normal tissues (Table 1). Most noticeably, PSAP is overexpressed and/or amplified in human prostate cancer tissues, xenografts, and cell lines. Quantitative SNP array hybridization in conjunction with southern hybridization and quantitative real-time PCR demonstrated a frequency of 20.6% for PSAP amplification (4 out of 25 prostate cancer xenografts and metastatic tissues and three out of nine prostate cancer cell lines). Expression of PSAP protein and mRNA in malignant prostate cancer cells is exclusively higher than normal prostate epithelial and stromal cells. Immunoblotting of conditioned media derived from prostate cancer cells shows the presence of PSAP-immunoreactive bands with approximate molecular size of 72-kDa, 140-kDa, and 220-kDa. It is not clear whether or not the 140- and 220-kDa bands represent the dimeric or trimeric form of PSAP. In addition, PSAP mRNA and protein expression is higher in several androgen-independent than the androgen-dependent prostate cancer cell lines. This finding suggests that PSAP expression might be under androgenic, steroid hormone regulation, or feedback control mediated by the hypothalamus-pituitary-gonadal neuroendocrine axis. The involvement of pertussis toxin-sensitive GPCR-dependent mechanism for in vitro biological activities of PSAP (or its active molecular derivatives such as saposin C, TX14A) has been demonstrated in a number of cell lines. In addition, using human and mouse fibroblasts and in vivo studies, it has been demonstrated that PSAP entry into the cells is also possible via at least three other independent receptor system including the mannose receptor, mannose-6-phosphate (M-6-P) receptor, and low density lipoprotein receptor-related protein (LRP). Cell type-specific distribution of any of the above receptor systems, their relative abundance, their involvement in various biological activities of soluble PSAP and/or saposin C (e.g., cell signaling, sphingolipid transport), or post-receptor occupancy events require additional studies. |
| Localisation | Prosaposin exists as a lysosomal, integral membrane, and an intracellular protein. In addition, prosaposin also exist as an integral membrane protein. The relative abundance of prosaposin is believed to be the highest as a secretory (soluble) protein and the lowest as an integral protein. However, it is not clear whether there is a tissue or cell type-specificity (e.g., benign versus malignant cells, epithelial versus stromal cells) for PSAP distribution. |
| Function | Prosaposin is a dual function molecule; as the precursor of intracellular lysosomal saposin proteins involved in sphingolipid hydrolysis activity and as a secreted soluble protein with neurotrophic activities, including growth, development, and maintenance of the peripheral and central nervous system, nerve regeneration and plasticity, stimulation of neurite outgrowth, stimulation of neuroblastoma cells proliferation, protection from cell-death or apoptosis, and activation of MAPK- and PI3K/Akt-signaling pathways. Column chromatography data indicated the formation of stable complexes between PSAP/saposins and several gangliosides. It has been suggested that PSAP functions as a sphingolipid binding protein and on the cell surface, complex formation between PSAP and gangliosides may suggests a role for this molecule in ganglioside function. Whether or not there is a link between the function of secreted soluble form as a trophic factor and its role as a ganglioside-binding or -career protein remain to be understood. Saposins function as coprotein for intracellular degradation of sphingolipids. Saposin A and C is involved in hydrlysis of glucosylceramide and galactosylceramide. saposin B stimulates galacto-cerebroside sulfate hydrolysis, GM1 ganglioside, and globotriaosylceramide. Saposin C is the activator of sphingomyelin phosphodiesterase. While several members of CD1 proteins are involved in lipid presentation to T cells, prosaposin-deficient mice exhibit certain defects in CD1d-mediated antigenic presentation suggesting that saposins are involved in mobilization of lipid monomers from the lysosomal membrane and their association with CD1d. In addition, prosaposin-deficient fibroblasts transfected with another member of CD1 family (CD1b) also failed to activate lipid-specific T lymphocytes. Upon reconstitution of fibroblasts with saposin C, T-cells response was restored. These findings might be suggestive of potential implications for saposin C or perhaps PSAP in recognition of tumor antigens. Several reports have identified a number of linear 5-22 amino acid segments called prosaptides (e.g., D5, TX14A) that demonstrate in vitro and/or in vivo neurotrophic activities. These bioactive sequences are located at the downstream region of saposin C domain of PSAP. Prosaptides, saposin C, or PSAP exert their effect at least partially, by binding to a single high-affinity G protein-coupled receptor. This receptor has been partially characterized but not cloned. In malignant cells and tissues, several classic reports have indicated a pluripotent regulatory role for saposin C and PSAP in prostate cancer with potential involvement in prostate carcinogenesis or progression toward metastatic or androgen-independent state. Immunohistochemical staining on benign and malignant prostate tissues revealed an intense cytosolic and anti-prosaposin immunoreactivity in tumor cells, stromal, endothelial, and inflammatory mononuclear cells and the intensity of staining was proportional to the overall Gleason's score. PSAP-immunoreactivity was also noticeable as extracellular deposition in hypercellular regions in high-grade prostatic tumors. In addition, PSAP and/or its active molecular derivatives (saposin C or TX14A) stimulate prostate cancer cells growth, motility, and invasion, upregulates uPA/uPAR expression, activates the p42/44 MAPK (Raf-MEK-ERK-RSK-Elk-1 signaling cascade), p38 MAPK, and SAPK/JNK family members of the MAPK superfamily and PI3K/Akt signaling pathways, and protects cells from apoptotic cell-death induction by etoposide via modulation of caspase-3, -7, and -9 expression/activity and/or the PI3K/Akt signaling pathway activation. |
| Homology | The four saposin A-D proteins share a great deal of homology (~50% ) in their amino acids sequences. In addition to these, saposins also contain 6 highly conserved cysteines. Considering all these structural similarities, they differ from each other for their specificity of intracellular or potential extracellular functions. Among fopur saposins, cross-species analysis of saposin sequences, show evolutionary conservation for saposin A, B, and D. However, with the exception to the neurotrophic sequence, saposin C sequence appear to be more species-specific. For example, from the linear human saposin C-neurotrophic sequence (LIDNNKTEKEILD): (1) LID-NK and TEKEIL is shared with RNA polymerase subunit of sheep Pox virus; (2) LINK and TEKEL is shared with Lumpy skin disease virus; (3) NNK and EKEIL is shared with the Hemagglutinin influenza A virus; (4) NNTEK-IL is shared with HIV-I envelop glycoprotein; (5) DN---EKEI is shared with Bacillus anthracis; or (6) LIDN-KT-KEI is shared with flagellar filament outer layer protein precursor (sheet protein) of Lyme disease spirochete.
Although these linearly ordered sequence homologies appear to be remote and partial, but due to the observed profound biological activities of the neurotrophic sequence-derived peptides (in in vitro and in vivo studies) and their relative hydrophilic nature, their presence in pathogenic agents (e.g., HIV virus, anthrax) might have some potential clinical application or might be useful in understanding the mechanism underlying their pathogenicity (with respect to eukaryotic cells). |
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