Alternative Splicing and Tissue Distribution of the Mouse Sulfated Glycoprotein-1 (SGP-1IProsaposin) mRNA and its Translation Product
Nina Hay
Department of Anatomy and Ce11 Biology McGill University Montreal, Quebec Canada November, 1996
A Thesis Submitted to the Faculty of Graduate Studies and Research in Partial Fulfilment of the Requirements for the Degree of Master of Science
O Nina Hay 1996 National Library Bibliothbque nationale B .canada du Canada Acquisitions and Acquisitions et Bibliographie Services services bibliographiques 395 Wellington Street 395, nie Wellington OttawaON K1AON4 Ottawa ON KtA ON4 Canada Canada
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The author retains ownership of the L'auteur conserve la propriété du copyright in this thesis. Neither the droit d'auteur qui protège cette thèse. thesis nor substantial extracts fiom it Ni la thèse ni des extraits substantiels may be printed or othewise de celle-ci ne doivent être imprimés reproduced without the author's ou autrement reproduits sans son permission. autorisation. Short Title: Alternative splicing of SGP- 1 rnRNA and its tissue distribution. Abstract
Sulfated glycoprotein- l/prosaposin exists in two forms. A 65 kDa protein which is the precursor of four lysosomal saposins A, B, C, and D, involved in giycosphingolipid degradation, and a 70 kDa extracellular protein present in several body fluids which has been thought to function as a lipid transfer protein. Human prosaposin has also recently been considered as a neurotrophic factor in the brain. The deficiency of saposins B and C result in two variant forms of lysosomal storage disorders; metachromatic leukodystrophy and Gaucher's disease, respectively. No deficiencies have been reported for saposins A and D. The alternative splicing of the prosaposin gene results in the inclusion or exclusion of a 9 bp insertion of exon 8, which is located in the saposin B domain of this gene. Thus, exon 8 codes for three arnino acid residues (Gln-Asp-Gln), which may potentially be present or absent in human prosaposin. Recently, it was show that the alternative splicing of the prosaposin gene may be tissue specific and that a possible function of the three amino acid insertion could be to alter the binding specificity of saposin B towards different glycosphingolipids. We have recently cloned the murine SGP-1 gene and found that it also contains îhe 9 bp exon 8. In the present study we have used reverse transcription-polymerase chain reaction (RT-PCR)to study the distribution of the altematively spliced mRNAs in several tissues. We have also used Northern Blot analysis to confirm the expression and stability of these transcripts, and light microscope immunocytochemistry to examine whether or not alternative splicing affects the translation of the rnRNA transcripts into mature proteins. The results showed that muscle, heart and brain contained the 9 bp insertion for exon 8, whereas testis, lung, pancreas, spleen, and kidney were devoid of the insertion. Sequencing of two DNA fragments from brain and kidney confirmed these findings. Furthermore, Northern Blot analysis showed that a stable SGP-1 mRNA transcript was present in dl the tissues despite alternative splicing. Immunoperoxidase staining with the anti SGP-1 antibody reveded that the mRNA forms were translated into a mature protein in al1 tissues examined. Therefore, it was concluded that alternative splicing of the SGP- 1 gene occurs in the mouse; that this mechanism appears to be tissue specific; and that both mRNA forms (with or without the 9 bp insertion of exon 8) result in stable RNA molecules which translate SGP- 1. These results suggest that alternative splicing may confer sphingolipid specificity to saposin B. Résumé
La glycoprotéine sulfatée 1, SGP- llprasaposine existe sous deux formes: une molécule de 65 kDa qui donne naissance à quatre saposines lysosomiales (A-D) impliquées dans la dégradation des glycosphingolipides; et une protéine de 70 kDa présente dans certaines secrétions et qui semble jouer un rôle dans le transport des lipides. La prosaposine humaine est aussi considérée comme un facteur neurotrophique du cerveau. Une déficience en saposines B et C ont respectivement pour conséquence deux formes de pathologies de nature lysosorniale: la leukodystrophic métachromatique et la maladie Gaucher. On n'a pas encore démontré les conséquences pathologiques d'une absence des saposines A et D. L'épiçage alternatif du gène responsable de la prosaposine résulte dans l'inclusion ou l'exclusion de 9 bp de l'exon 8 qui est localisé dans la région saposine B de ce gène. L'exon 8 sert de code pour trois acides aminés (Gln-Asp-Gin) qui peuvent donc être présents ou absents de la prosaposine humaine. Il a été indiqué récemment que I'épiçage alternatif du gène prosaposine doit être tissue-spécifique et qu'une fonction potentielle des trois acides aminés serait de déterminer la liaison de la saposine B avec certaines glycosphingolipides. Nous avons récemment cloné le gène SGP- 1 de souris et observé la présence des 9 bp de l'exon 8 dans ce gène. Au cours de la présente étude nous avons utilisé la méthode RT-PCR (reverse transcription-polyrnerase chah reaction), pour étudier la distribution des mRNA épicés alternativement dans plusieurs tissues ou organs. Nous avons également utilisé la méthode Northem BIot pour confirmer l'expression et la stabilité des transcriptions et l'immunocytochimie en microscopie optique pour déterminer si oui ou non l'épiçage alternatif afTecte la traduction des mRNA. Nos résultats démontrent que le muscle, le coeur et le cerveau contiennent l'insertion des 9 bp de l'exon 8, tandis que le testicule, poumon, pancreas, rate et rein ne contiennent pas cette insertion. Le séquançage de deux fragments de DNA du cerveau et du rein ont confirmé ces résultats. De plus l'analyse par Northem Blot a montré que le rnRNA-SGP-1 traduit est stable et présent dans tous les tissues examinés et cela sans eRet de I'épiçage alternatif L'immunocytochimie à l'aide d'un anticorps anti-SGP-1 marqué par la peroxidase, révèle que le mRNA est traduit en protéines matures dans tous les tissues analysés. Nous concluons donc que I'épiçage alternatif du gène SGP- 1 existe bien chez la souris, que ce processus semble être tissues-spécifique, et enfin que les deux formes de mRNA (avec ou sans l'insertion de 9 bp de l'exon 8) produites sont stables et donnent naissance a la SGP-1. Ces résultats suggèrent que I'épiçage alternatif peut conférer à la sapsine B un mode d'action spécifique sur les sphingolipides présents dans les divers tissues. To Kevin, Mom, Dad, and Symon First and foremost, 1 would like to thank my supervisor, Dr. C.Morales, for his kindness, patience, knowledge, and advice. He was never too busy to help in both my experiments as well as the writing of this thesis. His supervision was valuable and greatly appreciated.
To Dr. El-Alfy, who was always available for questions, or just to chat, his advice, sense of humour and genuine kindness made this year a pleasure.
To Dr. Y. Clermont for his enthusiasm and encouragement. 1 also thank him for translating the abstract for this thesis.
To Qing Zhao, who was my right hand in al1 my work. She is the reason that this project was able to develop, and 1thank her with all my heart. Her cheefilness, advice, and knowledse was an inspiration to me.
To Farkhondeh Pouresmaeily, whose words of advice and continuous encouragement kept me going despite the struggles.
To Maire Leyne, my lab cornpanion, who was always there when 1 needed a lift, 1 am thankhl for her suppon, and her wonderful friendship.
To Dr. Suleiman lgdoura for his sound advice and help throughout this project, and for always making me Iaugh.
To al1 the graduate students and staffmembers whom 1 became close with, you know who you are, thank you for making this year a mernorable one.
To Adam Obidniak for his patience and technicd work on this project.
To my family and fi-iends who put up with me through al1 my endeavours, 1 could not have done this without their continuous encouragement. Finally, to Kevin, for his guidance, support, confidence, and love, which will never be forgotten.
This research was supported by an M.R.C. grant to Dr. Carlos R. Morales.
vii Abbreviations
P-ME: beta-mercaptoethanol bp: base pair(s) BSA: bovine serum albumin cDNA: complementary deoxyribonucleic acids dd H,O: double distilled water DEPC: diethyl pyrocarbonate dNTP: equimolar of dATP, dCTP, dGTP, and dTTP E.Coli: Escherichia coli EDTA: ethylenediarninetetraacetic acid ER: endoplasmic reticulum GSL: glycosphingolipids IPTG: isopropyl beta-D-thiogalactopyranoside Kb: kilobase pair(s) ma: kilodalton(s) ml: millilitre(s) mM: milimolar mRNA: messenger ribonucleic acid NaCI: sodium chloride oligo: oligonucleotide(s) PCR: polymerase chain reaction RT-PCR: reverse transcription-yolymerase chain reaction SAP: sphingolipid activator protein SAPLIP: saposin-like proteins SDS: sodium dodecyl sulfate SC P- 1: sulfated glycoprotein- l snRNP: small nuclear ribonucleoproteins TBS: tris-base buffered saline UV: ultraviolet pl: micro litre Table of Contents
... Abstract ...... 111 Résumé ...... iv Dedication ...... vi Acknowledgements ...... vii ... Abbreviations ...... vrii
1. Introduction ...... ,...... 1
II. Review of the Literature ...... 3
Glycosphingolipids ...... 3 A) Glycosphingolipid (GSL) Structure. Distribution and Function ...... 3 B) Glycosphingolipid Biosynthesis ...... 3 C) Lysosomal Degradation of Glycosphingolipids ...... 4
Prosaposin and Saposins ...... 5 A) Historical Background ...... 5 B) Prosaposin Structure. Targetinç and Function ...... 6 C) Saposins Structure and Function ...... 9 D) Structure and Evolution of the Prosaposin Gene ...... 14 E) Saposins and Lysosomal Storage Diseases ...... 15 F) Alternative Splicing of Prosaposin rnRNA ...... 18
3. Saposin-Like Proteins (SAPLIPs) ...... 20
4 . General Mechanism of Alternative Splicing ...... 21 A) RNA Synthesis ...... 21 B) RNA Splicing ...... 22 C) Alternative RNA Splicing ...... 23 5. Objective of the Present Research ...... 24
III. DIAGRAMS ...... 25
IV .Materials and Methods ...... 29
1. Total RNA Extraction ...... 29 A) Preparation of Solutions ...... 29 B) Total RNA Isola.tion and Purification ...... 29
Analysis of Total RNA Using the Polymerase Chain Reaction ...... 30 A) Designing PCR Primers ...... 30 B) RT-PCR ...... 31 C) Polymerase Chain Reaction (PCR)...... 32 D) Analysis of PCR Products ...... 33 E) Isolation of the DNA Fragments Amplified by PCR ...... 34
Cloning of the PCR Products ...... 34 A) Ligation of the PCR product into a pGEM@-TVector ...... 34 B) Transformation of Ligated PCR:pGEMm-TVector into High Efficiency Competenr Cells ...... 35 C) Identification of the Positive Clones ...... 35 D) Isolation of Recombinant Plasmid DNA ...... 36
4 . Sequencing of the PCR Products ...... 36 A) Preparing Plasmid DNA for Primer Anneding ...... 36 B) Sequencing Reactions ...... 37 C) Sequencing Gel ...... 37
5. Analysis of RNA using Northern Blot Hybridization ...... 38 A) Agarose/Forrnaldehyde Gel Electrophoresis ...... 38 B) Transfer of RNA from Gel to Membrane ...... 38 C) Preparation of the RNA Probe ...... 39 D) Hybridization Analysis ...... 40 Analysis of the Translated Product of the SGP-1 mRNA Using Light Microscopy Immunocytochemistry ...... 40 A) Tissue Preparation ...... 40 B) Immunocytochemistry using Immunoperoxidase Staining ...... 41 C) Immunocytochemicai Controls ...... 42
V . Results ......
Identification and Analysis of the Tissue Specific Alternative Splicing of the SGP-1 mRNA in the Mouse ...... 43 A) Amplification of Two DNA Fragments using PCR ...... 43 B) Cloning of the PCR Products ...... 44 C) Identification of the Positive Clones ...... 45 D) Sequencing of the PCR Products ...... 45
Analysis of the mRNA Expression of the SGP-1 Gene ...... 46
Distribution of the Translated Product of the SGP-1mRNA ...... 46 A) CortexoftheBrain ...... 46 B) Choroid Plexus ...... 46 C) Heart ...... 46 D) Musde ...... 46 E) Spleen ...... 47 F) Lung ...... 47 G) Pancreas ...... 47 H) Kidney ...... 47 1-J) Testis ...... 47 K) Testis of the Rat- Positive and Negative Control ...... 47 VI1. Discussion ......
1. Identification and Analysis of the Tissue Specific Alternative Splicing of the SGP-1 mRNA in the Mouse ...... 59 A) Amplification of Two DNA Fragments Using the Polymerase Chain Reaction ...... 59 B) Cloning of the two DNA Fragments ...... 60 C) Sequencing of the Two DNA Fragments ...... 61
2 . Regional Distribution of the SGP-1 mRNA ...... 62
3. Regional and Cellular Localization of the Translated Product of the SGP-1 mRNA ...... 63
VI11 . Conclusions ......
IX . clrieinal Contributions ...... 66
X . References ...... 67
xii 1. Introduction
Prosaposin (SGP-1) exists in two forms, an intracellular form of 65 kDa which serves as a lysosomal precursor that is proteolytically processed into four saposins A, B, C, and D, and a 70 kDa extracellular, unprocessed form which is present in several body fluids, and has been thought to function as a transfer protein for glycosphingolipids, as well as a neurotrophic factor in the brain (Kishimoto et al., 1991, O'Bnen et al., 1988, Fürst and Sandhoff, 1992). Saposins contain similar amino acid sequences, and conserved positions of cysteines and asparagines. They are heat stable and resistant to protease activity, and therefore contain highly ordered secondaiy structures (O'Brien and Kishimoto, 1991, O'Brien et al., 1988, Fürst and Sandhoff, 1992, Stevens et al, 1993, and Kishimoto et al., 1992). Despite these sirnilarities in their structures, the functional properties of saposin B differ from those of the other three saposins (Kishimoto et al., 1992). Saposin B is thought to bind to glycosphingolipid (GSL) substrates while the other saposin proteins seem to interact with the lysosomal hydrolases (O'Brien and Kishimoto, 1991). Al1 four saposin proteins, however, are considered to be cofactors in the degradation of GSLs by acid hydrolases. The saposins and prosaposin have also been implicated in having the ability to act as lipid binding and transfer proteins for GSLs (Vogel et al.. 1991 and Hiraiwa et al., 1992). Alternative splicing of the prosaposin gene leads to the inclusion or exclusion of a 9 base pair (bp) or 6 bp insertion which is otherwise known as exon 8. This exon is located within the saposin B domain of the gene and codes for either three (Gin-Asp-Gln) or two (Asp-Gln) amino acids (Holtschmidt et al., 1990). The three prosaposin mRNAs that could result from this altered splicing have been found to be present in both normal humans, as well as in patients who have been diagnosed with a variant fom of metachromatic leukodystrophy (Zhang et al., 1990). The fùnction of this exon 8 insertion, however, has not been fully clarified. Lt has been postulated that this 9 or 6 base pair insertion may function in altering the binding specificity of saposin B towards different glycosphingolipids within various tissues. Further research is therefore crucial in order to determine the actual fùnction of this exon, and to understand the mechanisms involved in the alternative splicing of the prosaposin gene. Our laboratory has receotly cloned the prosaposid sulfated glycoprotein- 1 (SGP- 1) gene in the mouse, and found exon 8 to be part of it. Based on this finding, the first objective of this thesis was to detemine whether the alternative splicing of the SGP- I/prosaposin gene occurs in the mouse, and is this mechanism tissue specific. The second objective was to examine the SGP-I mRNA expression within twelve different tissues of the mouse, and to determine whether or not the alternative splicing of this gene affects the transcription and stability of the mRNA transcript, using Northern Blot analysis. Finally, the third objective was to confirm whether or not the altematively spliced SGP- I mRNAs of the mouse are translated into a mature protein, by investigating its tissue distribution at the light microscopic level, using an anti- SGP- I antibody. In the following section, a review of the literature will examine the importance of the SGP-l/prosaposin gene and its four mature saposin products with regard to their structures, fùnctions, and physiological roles in several human diseases. It will also focus on the research that has been done on the alternative splicing of prosrposin mRNA, how it occurs, and the proposed function for exon 8. II. Review of the Literature
1. Glycosphingolipids A) Glycosphingolipid (GSL) Structure, Distribution and Fu nction Glycosphingolipids (GSLs) are components of the plasma membrane in al1 eukaryotic cells. They contain a hydrophobic ceramide moiety, which anchors them to the cell membrane, and a hydrophilic extracellular oligosaccharide chain. DifTerences in the number, type, and linkage of sugar residues in the oligosacchande chain create the broad range of GSLs that exists (Hakomori, 198 1). They are also assembled in cell-type-specific pattems at the cell surface, and these patterns are continuously changing according to the situation of the ce11 (Sandhoff and Kolter, 1996). GSLs are thought to be involved in ce11 differentiation and morphogenesis, they are implicated in the interaction with vimses, toxins and bacteria at the cell surface, they interact with membrane bound receptors and enzymes, and finally, they form a protective layer together with membrane glycoproteins and proteoglycans on biological membranes, preventing them from inappropriate degradation (van Echten and Sandhoff, 1993, Sandhoff and Kolter, 1996).
B) Glycosphingolipid Riosy nthesis The biosynthesis of sphingolipids takes place in the endoplasmic reticulum (ER) and the Golgi apparatus much like the biosynthesis of glycoproteins (Sandhoff and Kolter, 1996, Schwarzmann and Sandhoff, 1990). Although the biosynthesis of N-linked glycoproteins and their mechanisms of transport have been well documented, the site of glycosphingolipid biosynthesis and their movement along the transport pathway is still not fùlly clarified. The initial steps of sphingolipid biosynthesis involving the formation of ceramide is thought to take place at the cytosolic face of the ER, and is catalysed by several membrane bound enzymes (Sandhoff and Kolter, 1996, van Echten and Sandhoff, 1993). Ceramide is then modified by the addition of a phosphorylcholine moiety to form sphingomyelin or by stepwise glycosylation, to form glycosphingolipids. The formation of sphingomyelin occurs at the luminal surface of the Golgi apparatus, whereas the synthesis of the glycosphingolipids begins at the cytosolic face where the enzymes (glucosyltransferase and galactosyltransferase) catalysing the first two glycosylation steps are available (Sandhoff and Kolter, 1996). Following these steps, the sequential addition of monosaccharides and sialic acid residues takes place on the growing oligosaccharide chain, creating more complex GSLs (Hakomori, 198 1). These sugar additions are catalysed by membrane bound glycosyltransferases located at the luminal surface of the Golgi apparatus (van Echten and Sandhoff, 1993). The GSL products are then transported to the plasma membrane via exocytosis (SandhofT and Kolter, 1996).
C) Z,ysosonzal Degrathtion of Glycosphingolipids The conventional model for the degradation of plasma membrane components is a process whereby the membrane components are endocytosed, and reach the lysosomes by traversing early and late endosornes (Griffiths et al., 1988). This model is dificult to apply to the degradation of glycosphingolipids, because the outer leaflet of the lysosomal membrane is covered with a thick glycocalix composed of material that is similar to glycosphingolipids. Thus, the seleclive degradation of the glycosphingolipids within the lysosomal cornpartment would be difficult to achieve without the lysosomal membrane also undergoing degradation. In an alternative model, the GSLs originating fi-om the outer leaflet of the plasma membrane fom intraendosomal vesicles (also referred to as multivesicular bodies) by budding off from the endosomal membrane into the endosomal lumen. These intraendosomal vesicles subsequently become intralysosomal vesicles when they reach the lysosomes (Sandhoff and Kolter, 1996). This pathway ensures that the GSLs that are destined for degradation enter the lysosof and face the lysosomal hydrolases, while the outer lysosornal membrane components remain intact and do not undergo degradation (van Echten and Sandhoff, 1990). The glycosphingolipids are then degraded by the stepwise action of these specific acid hydrolases within the lysosol (Schwarzmann and Sandhoff, 1990). Evidence that supports this alternative pathway is described by Harzer et al. (1989), who observed that there is an accumulation of multivesicular storage bodies in the Kupffer cells of patients with a deficiency in the prosaposin protein, and that there is a deposition of storage material in the lysosomal lumen in the case of a deficiency in glycolipid degradation. These observations support the assumption that the GSLs are incorporated into intraendosomal and intralysosomal vesicles for degradation. Those GSLs that contain a hydrophobic head group extending far enough into the extracellular space are directly attacked by the acid hydrolases, whereas the GSLs that contain short oligosacchande chains (3 or less monosaccharide residues), require a cofactor or sphingolipid activator to help the acid hydrolase degrade the GSL (Sandhoff and Kolter, 1996). These sphingolipid activator proteins (SAPs or saposins) are described in detail in the following sections.
2. Prosaposin and Saposins A) Historica B~nckground The first saposin (saposin B or SAP 1) was described as being a heat stable protein that is required for the hydrolysis of sulfatides by arylsulfatase A (Fischer and Jatzkewitz, 1977). The second saposin (saposin C or SAP 2) was discovered by Ho and O'Brien ( 197 1), and was thought to stimulate the hydrolysis of glucosylceramidase. When the cDNA encoding saposin B (or SAP 1) was cloned (Dewji et al., 1986) its sequence showed marked similarity to a segment of a rat protein in the testis, sulfated glycoprotein-1 (SGP-1) (Collard et al., 1988). This discovery led investigators to determine the sequence of the human cDNA encoding the saposin C precursor, and it was found that the precursor gene encoded both saposin B (SAP 1) and saposin C (SAP 2). as well as two other sirnilar domains (O'Brien et al., 1988). The investigators concluded that SAP 1 and SAP 2 and perhaps two other SAP-like proteins are generated by the proteolytic processing of the same large rnolecular weight precursor (SAP precursor or prosaposin) (O'Brien et al., 1988). Meanwhile, Collard et al. (1988) reported cloning of the cDNA for the SGP-1 protein secreted by rat Sertoli cells. SGP-1 was found to contain a similar sequence to the human SAP precursor except for an additional prohe nch sequence coding for 3 1 amino acids located between SAP-like regions 3 and 4 of the SGP-1 cDNA (Collard et al., 1988). Collard et al. (1988), also found that the four domains in the SGP-1 protein are highly homologous to SAP 1 and SAP 2 in the human. Following these findings, a third protein named component C (identical to saposin D) was isolated by Monmoto et al. (1988), and soon after, a fourth protein was isolated by the same investigators, and is now called saposin A. The nomenclature of saposins A-D was proposed in order to avoid confusion with the previous terms that were used to identify these proteins. The sphingolipid activator proteins (SAPs) were therefore named saposin A, B, C and D based on the placement of these four domains in the prosaposin precursor (Diagram l), and to emphasize the fact that they were al\ derived from the same genetic origin (Morimoto et al., 1988).
B) Prosaposin Sîructure, Tnrgeting and Function S'tr~~ctrrre Prosaposin is recognized as the lysosomal precursor of four sphingolipids activator proteins narned saposin A, B, C and D. The prosaposin cDNA encodes a polypeptide chain of 524 amino acids starting with a signal peptide of 16 amino acids, which allows the protein to enter the endoplasmic reticulum (ER) (Nakano et al., 1989). This protein is thought to be generated by the CO-translationalglycosylation of a 53 kDa polypeptide at five gl ycosyiation sites (Diagram 1). The protein is then fùrther glycosylated in the Golgi apparatus to a 70 kDa protein which is either secreted into the extracellular space, or transported to the lysosomal cornpartment and proteolytically cleaved into 10-1 5 kDa polypeptides (saposins) (O'Brien and Kishimoto, 1991). At the junction of each saposin domain within the prosaposin protein, there are dibasic amino acids that are potential proteolytic cleavage sites (seven lysines and one arginine) (O'Brien and Kishimoto, 1991). It is not known which cleavage sites are processed first, but intermediate cleavage products (intermediates between prosaposin and mature saposins) have been detected using immunoblot experiments in human and rat tissues (O'Brien et al., 1988, Sano et al., 1989, Hiraiwa et al., 1993). Hiraiwa et al. (1993) have found these intermediate cleavage products while purifying prosaposin from seminal plasma, indicating a possible proteolytic pathway for prosaposin. These investigators have demonstrated through immunochernical experimentq that the first cleavage product, trisaposin, contains the domairis B, C, and D, and must result from cleavage between the domains A and B. They also have detected a second intermediate cleavage product, disaposin, which contains domains for saposins C and D. Thus, these experiments suggest that the proteolysis of prosaposin in human testis occurs by the removal of one domain at a time from the N-terminal end of this protein (Hiraiwa et al., 1993). Whether this method of proteolytic processing occurs in al1 tissues and cell types remains to be elucidated.
Targeting Rat sulfated glycoprotein (SGP-1) is homologous to the human prosaposin (Collard et al., 1988). SGP- 1 is synthesized in the Sertoli cells of the rat as a 65 kDa precursor protein which is post-translationally glycosylated to a 70 kDa fom, and then secreted into the extracellular space (Fürst and Sandhoff, 1992). The 3 1 amino acid proline rich segment which is absent in the human prosaposin, has been implicated in this secretory routing of SGP-I to the extracellular space (O'Brien et al., 1988). Although SGP-1 was originally thought to be exclusively secreted to the extracellular space, recent irnmunocytochemical studies have also localized the 65 kDa form of SGP-1 to the lysosomes of Sertoli cells (Igdoura et al., 1993). In a study by lgdoura and Morales (1 995)' reverse-phase HPLC revealed that 15 kDa polypeptides, that correspond to the human saposins, are also present in the lysosomal compartments of rat Sertoli cells. This suggested that SGP-1 may also be proteolytically processed in the lysosomes as is hurnan prosaposin. Thus, it is apparent that the 65 kDa form of SGP-1 is the precursor for both the 70 kDa secreted protein and the saposins that are processed in the lysosomes of Sertoli cells (Igdoura et al., 1993). The 65 kDa form of rat SGP-1 is thought to be delivered to the lysosomal compartment directly from the Golgi apparatus, while the 70 kDa form of SGP- 1 is released to the extracellular space and does not seem to enter the endocytic compartment of Sertoli cells (Igdoura and Morales, 1995). These investigators provide evidence from immunochemical experiments, indicating that the 70 kDa protein was not the predominant form in the lysosomes. In addition, their data revealed that the 65 kDa form of SGP- 1 was directed to the lysosomes in a mannose 6-phosphate independent manner (Morales et al.. 1996). The different targeting pathways of these two foms of SGP- 1 is potentially indicative of t he different functions that are associated with t his protein. fiitcriort Prosaposin or SGP-1 appears to have a few more functions in addition to servinç as a lysosomal precursor for the mature saposins (O'Brien and Kishimoto, 1991). The secretory form of prosaposin has been detected in human secretory fluids such as milk, cerebrospinal fluid, seminal plasma, pancreatic juice, and bile (Hineno et al., 1991). Furthennore, rat SGP-1, which is homologous to both human and murine prosaposin (Morales et al., 1995), is secreted into the seminiferous tubule by Sertoli cells (Collard et al., 1988). Hiraiwa et al. (1 991) demonstrated that prosaposin possesses binding and transfer properties and they postulated that the secretory form of prosaposin might be involved in the transfer of GSLs to the plasma membrane. Similarly. SGP-1 has been seen to interact with the plasma membrane of maturing spermatidg and to help in the transfer of membrane lipids during their development (Morales et al., 1995, Hiraiwa et al., 1991). Thus, SGP-I or prosaposin has been implicated in fùnctioning as a glycolipid binding and transfer protein (Collard et al., 1988). Prosaposin/SGP-1 has also been identified as a possible neurotrophic factor (O'Brien et al., 1994). Prosaposin is present in human and rat brain as an unprocessed protein (O'Brien et al., 1988, Sano et al., 1989). Prosaposin has also been localized in the neurons of rat and human, in high concentrations. O'Brien et al. (1994), report that prosaposin is a neurotrophic factor that induces the differentiation of neurons. They also have detemined t hat the reçion for this function is located in the saposin C domain of prosaposin. Their results indicate that when neuronal cells are treated with saposin C, it stimulates the phosphorylation of several proteins involved in the signal transduction cascade (O'Brien et al., 1994). In addition, immunolocalization studies have been done using an anti-prosaposin antibody, which show high reactivity in the motor neurons of humans and adult rats, as well as in the nervous system of the developing rat (Sano et al., 1989). Sano et al. (1994) have also demonstrated that prosaposin functions as a neuroprotective and regenerative agent in vivo. The role of prosaposin as a neurotrophic factor, however, is a recent finding and further work must be done in order to completely understand its functional significance in the nervous system. The distribution of prosaposidSGP-1 and its mature saposins varies according to the tissues being examined. O'Brien et al. (1988) describe prosaposin to be in its highest concentration in brain and testis, followed by kidney, spleen, and liver. Sano et al., (1989) have also investigated the distribution of saposins and prosaposin in rat tissues and have found that saposins are dominantly expressed in spleen, lung, liver, and kidney, whereas skeletal muscle, hean, and brain rnainly contain the prosaposin precursor. Prosaposin has also been found to predominate in plasma, and its presence tends to increase during brain development (Sano et al., 1989). In addition, the mRNA of prosaposin is abundant in brain during development and can be visualized in neurons after immunostaining with an anti-prosaposin antibody (Kondoh et al., 1 9%). Thus, prosaposin/SGP- 1 is possibly a multi functional protein.
C) Saposins Structure and Function Sapusirr A Saposin A is an 84 amino acid protein located at the N-terminal domain of the prosaposin molecule. It has a molecular weight of 16 kDa before glycosylation, two giycosylation sites, and contains six cysteine residues, al1 in disulfide linkage (Diagrams 1-2) (Kishimoto et al., 1992). It acts as a cofactor for glucosylceramide P-glucosidase activity by causing a conformational change in the enzyme, that increases enzyme activity (Fabbro and Grabowsky, 1991). Saposin A binds to P-glucosidase at a separate site fiorn where the acid lipids bind (Morimoto et al., 1990). Although saposin A is a major cofactor, the degree of stimulation that it has on this enzyme is oniy one third compared to that of saposin C (Kishimoto et al., 1992). In addition to its capacity to activate P-giucosidase, saposin A can also stimulate galactosylceramidase (Fürst and Sandhoff,1 992). Sapsiin B Saposin B was the first saposin to be identified (Fischer and Jatzkewitz 1977). It consists of 80 amino acids, and has a molecular mass of 8-1 1 kDa before glycosylation (Fürst and Sandhoff, 1992). Saposin B, unlike the other saposins, does not bind to the enzymes (Kishimoto et al., 1992). Instead, it interacts with lipid substrates and solubilizes them so that they can be hydrolysed by lysosomal enzymes (Vogel et al., 1991, Fischer and Jatzkewitz, 1978). Saposin B extracts the glycosphingolipid molecule fiom the micelles or membranes by forming a water soluble complex (Vogel et al., 1991). Enzymes can then cleave these complexes, and catalyse the degradation of the GSL (Vogel et al., 1991). It has been well documented that saposin B has a broad specificity for certain glycosphingolipids, and can stimulate the hydrolysis of GMl ganglioside, galactocerebroside sulfate, and globotriosylceramide (Wenger and Inui, 1984). The enzymes responsible for the GSL hydrolysis only recognize the substrate when it is bound to saposin B, but cannot recognize isolated substrate or saposin B. Thus, saposin B seems to act as a physiological detergent with a broad specificity that functions in solubilizing glycolipid substrates (Fürst and Sandhoff, 1992). A deficiency of saposin B leads to a variant form of a disease called metachromatic leukodystrophy. This deficiency causes the accumulation of cerebroside sulfate in the tissue of patients, and is transmitted as an autosomal recessive trait (Kretz et al., 1990).
Suposir, C Saposin C consists of an 80 amino acid polypeptide chain with a molecular mass of 5- 12 kDa depending on the degree of glycosylation. It also has six cysteine residues and forms disulfide bridges (Diagram 2) (Fürst and Sandhoff, 1992). Saposin C appears to be a cofactor in stimulating the hydrolysis of glucocerebroside by glucosylceramidase and galactocerebroside by galactosylceramidase (Kishimoto et al.. 1992). Saposin C does not seem to interact with the lipid substrate, but instead it binds to the enzyme to increase its catalytic capacity (Kishimoto et al., 1992). Through kinetic experiments, it has been demonstrated that saposin C binds to the enzyme at a separate site from where the acid lipids bind (Basu and Glew, 1985). These investigators showed that saposin C activates the enzyme glucosylceramidase, by further lowering the Km and raising the Vmax values. From this data, it was suggested that glucosylceramidase binds to acidic phospholipids of lysosomal membranes, causing a conformational change in the enzyme, and leading to the formation of aggregates, which in tum activates the enzyme. Saposin C then binds to this enzyme at a different site and causes the enzyme to shift into its most active conformation, thus creating a synergistic effect between saposin C and the acid lipid (Fürst and Sandhoff, 1992). More recently investigators have identified that it is the amino terminal end of the saposin C protein, consisting of 12 amino acids, that mediates the neurotrophic effect s of the prosaposin molecule. In addition, this region of the saposin C protein differs From the carboxyl-terminal area which is responsible for the binding of glucocerebrosidase (O'Brien et al., 1994). Glucosylceramide is a storage compound in a lysosomal storage disease called Gaucher disease. When there is a deficiency of saposin C or a mutation in the saposin C domain of the prosaposin gene, a fom of this disease occurs due to the lack of stimulation of the enzyme glucosylceramidase, which cannot hydrolyse its lipid substrate (Fürst and Sandhoff, 1992).
Sapclsir~B Saposin D was the last saposin protein to be identified along with saposin A. Its function is still poorly understood. It is located on the fourth domain of the prosaposin molecule, closest to the C-terminus (Kishimoto et al., 1992) (Diagram 1 ). Saposin D contains 78 amino acids, carries one N-linked carbohydrate chain, and has a molecular mass of 10 kDa (Fürst and Sandhoff, 1992 ). lt was initially thought to stimulate the degradation of sphingomyelin by sphingomyelinase (Fürst and Sandhoff, 1WZ), however, a çenetic disease caused by the complete absence of prosaposin was discovered to have normal sphinsomyelinase activity (Brodova et al., 1993). Clearly fùrther research on this protein is needed to identifi its actual fhction. Biochemicul Stmcttrre With the use of dichroism spectroscopy, al1 four saposins were found to have highly ordered secondary structures. Hypothetical analyses of these structures, using helical models as an aid. have been usefùl in elucidating the potential function of these proteins. Saposins 4 C, and D contain a high a-helical structure (4 1-53%), whereas saposin B contains a lower a-helical content (26%), and a much higher P-sheet (34%) (Morimoto et al., 1990). The helical contents were found to be the highest when the saposins were in their most active state. The hypothetical localization of a-helical, P-sheet, and p-turn regions in each saposin, using Chou-Fasman predictive mles (Chou and Fasman, 1978), suggests that there are similatities between saposin A, C, and D and a different structure for saposin B (O'Brien and Kishimoto, 1991). The model fcr saposin A and C indicates that they have helical structures closely resembling each other consisting of three a-helical columns (at 6- 16, 22-32, and 42-49 amino acid residues) which are flanked by P-turn regions (O'Brien and Kishimoto, 199 1). The model for saposin D indicates that it has structural similarity to A and C but differs in the region of the 42-62 arnino acid residues (O'Brien and Kishimoto, 199 1). Two models have been predicted for the structure of saposin B. The first model is unique in that it contains a large P-sheet region fiom amino acid 1-24, and a long COOH- terminal helical reçion (O'Brien and Kishimoto, 1991 ). This model consists of three cl-helices which are stabilized by interna1 disulfide cross-linking (O'Brien et al., 1988). These cross linkages are thought to maintain the stability of saposin B in conditions of extreme pH, heat, and the presence of proteinases (Fürst and Sandhoff, 1992). The second model for saposin B was developed by Stevens et al. (1 993), based on the presence of three intemal disulfide bonds discovered using mass spectrometry. The model contains four cc-helices and is similar to the first model in that each a-helical segment of 18 amino acids has an arnphipathic structure. The hydrophobic sides of each a-helix are facing one another, whereas the hydrophilic parts of the a-helical segments are exposed to the exterior of the protein. This theoretical structure is consistent with the water solubility of saposin B and its ability to bind to water-insoluble glycosphingolipids (Stevens et al., 1993). Both these models suggest that saposin B has a very ordered and stable secondas, and tertiary structure, with a hydrophobie centre or pocket that is presumed to bind lipids, and a disulfide cross-linking pattern that stabilizes the interaction of the helices (Stevens et al., 1993). Attempts have been made to correlate these two models of saposin B structure to the structures of saposins A, C, and Li, since al1 four of these proteins have sequence similarity, a similar alignment of their cysteine residues, and contain glycosylation sites (Diagrams l&2) (O'Brien and Kishimoto, 1991). Thus, saposins A, C, and D may have disulfide bridges, and secondary and tertiary structures analogous to saposin B (Stevens et al., 1993).
Other Fw~ctionsAttributed to Saposii~s ln addition to their role in the degradation of glycosphingolipids, saposins have also been irnplicated as glycosphingolipid binding and transfer proteins. As discussed above, Fischer and Jatzkewitz (1978) have demonstrated that sulfatides are extracted fiom micelles or membranes by saposin B, to give fieely water soluble activator-lipid complexes. According to Vogel et al. (1 99 l), this interaction should be reversible. They suggest that the sulfatide could be inserted into a different membrane fiom which it was extracted. Thus, in the absence of a hydrolytic enzyme, Vogel et al. ( 199 1) believe that the activator protein should work as a sulfatide transfer protein. To test their hypothesis, they have studied the transfer of sulfatides and other GSLs from donor to acceptor liposomes, in the presence of an activator protein. These investiçators have found that the transfer of labelled GSLs is catalysed by saposin B. ln addition, they have found that the highest transfer rates are observed for GSLs with long oligosaccharide chains. Furthemore, using binding studies, they have demonstrated that the transfer rates decrease with shorter oligosaccharide chains (Vogel et al., 1991). Hiraiwa et al. (1 992) have performed a similar study by investigating the possibility that prosaposin and saposins transfer gangliosides fiom donor liposomes to erythrocyte ghost membranes. They have demonstrated that al1 four saposins do in fact bind and transfer GSLs in vivo, and it has been suggestcd that the amounts of GSL bound to these proteins in vivo, rnay depend on different factors including, the state and type of ganglioside, the pH, and the presence of other lipids and other binding proteins. Their data also indicates that the highest affinity of prosaposin and saposins A-D is for the a-series ganglitetraose-type gangliosides that have terminal sialic acid residues (Hiraiwa et al., 1992). They attribute the GSL binding capacity of the saposins to the proposed structural mode1 of these proteins, which suggests that each saposin consists of a compact, rigidly cross-linked disulfide-bridged polypeptide, containing a common motif or hydrophobie pocket that binds lipids (O'Brien and Kishimoto, 1991).
D) Sîructure and Evolution of the Prosapnsin Gene The gene encoding human prosaposin is located on the long amof chromosome 10 (1 O,,-& (hui et al., 1985). The prosaposin gene is at least 17 Kb long containing 14 exons (Holtschmidt et al., 1991). which are intempted by several introns: two in saposin A, three in saposin B, one in saposin C, and two in saposin D (Kishimoto et al., 1992). Cornparison of the human, rat, and mouse cDNAs for prosaposin or SGP-1, demonstrates extensive regions of homology, including the four-region structure for saposins A, B, C, and D (Diagram 1) (Collard et al., 1988, Rorman and Grabowski, 1989). The only major difference in the sequences is a 90 base pair region encoding 30 amino acids in the rat and mouse Sertoli ce11 SGP- 1 proteins, which is absent in the hurnan cDNA of prosaposin (Rorman et al., 1991, Morales et al., 1995). Since the mature saposins have a very high degree of amino acid simiiarity (70-95%), it has been postulated that these proteins have ansen through gene duplication fiom an ancestral gene (Rorman et al., 1991). These investigators suggest that the ancestral gene must have duplicated pior to the introduction of introns. Then, following the insertion of introns, the new gene was duplicated in tandem to produce the final order of saposins. A gene remangement which involved a double cross over between the introns subsequently occurred, resulting in the alignment of the intron positions which are seen in the amino acid sequences of the saposins (Rorman et al., 1991). E) Saposins and Lysosomal Storage Diseuses The deficiency of saposins has been crucial in defining the physiological fùnctions of these proteins. The first defect involving saposins in a human disease originated in a variant fonn of metachromatic leukodystrophy having normal arylsulfatase A activity, but a deficiency of saposin B (Stevens et al., 1981). Four different defects within the saposin B domain of the prosaposin gene have been reported for this type of metachromatic leukodystrophy. Firstly, a patient with saposin B deficiency showed a point mutation of C to T, which caused a threonine to isoleusine exchange (Kretz et al., 1990). This single base change generates a saposin B protein that is proteolytically sensitive. Kretz et al. (1990) proposed that the point mutation in this patient abolishes the glycosylation site in saposin B, and therefore exposes a proteolytic site (arginine) which is situated two residues from the amino- terminal side of the glycosylation site. As a result, proteolytic degradation is increased, and the sarne proteolytic lysosomal enzyme that cleaves prosaposin at dibasic residues within the saposin boundanes, could also cleave this mutant protein (O'Brien and Kishimoto, 1991). T hu s, t his study describes a patient that developed a metachromatic Ieukodystrophy-like disease due to a defective saposin B protein, that is eventually degraded by the lysosomal enzymes. An interesting observation in this patient was that the maturation and transport of the prosaposin precursor was not affected, which lead to the successful formation of mature saposins A, C, and D (Kretz et al., 1990) A second genetic defect associated with saposin B deficiency was described by Zhang et al. (1990) who found an I l-ariiino acid insertion due to a C to A transversion in the middle of a 4 Kb intron of the prosaposin gene. This mutation leads to the activation of a new splice site that results in the insertion of 33 base pairs to the saposin B domain. Zhang et al. (1990) examined the sequence surroundhg the 33 bp insertion and found no other mutations, thus indicating that the insertion is probably the cause of the deficiency of saposin B. As in the previous patient described above, this defect only affected the stability of the mature saposin B and not the transport and processing of t he prosaposin precursor (Henseler et al., 1996). Thus, it was speculated that the il arnino acids, coded for by this insertion, may cause a change in the hydrophobic properties of saposin B, which could eventually lead to a decrease in its stability resulting in its degradation (Zhang et al., 1991). A third example of saposin B deficiency is a nucleotide transversion from G to C, causing a substitution of a cysteine for a serine in the mature protein (Holtschmidt et al., 1991). This nucleotide transversion may affect the protein's ability to form normal disulfide bonds because of the rnissing cysteine residue, resulting in the disruption of the normal three dimensional structure of this protein. This mutant protein subsequently becornes unstable and degrades, suggesting that the cysteine residues are very important for the stability of the mature saposins (Henseler et al., 1996). Finally, Henseler et al. (1 996) reported the presence of a saposin precursor with a 2 1 base pair deletion in the cultured fibroblasts of a patient. A mutation in an intron caused the activation of a splice site in exon 6, leading to the deletion of the first 21 base pairs of this exon. The resulting mutant mRNA codes for a prosaposin protein lacking 7 amino acids, 5 of which are from the N-terminus of the mature saposin B. Despite this mutation, the precursor protein is processed and iransported normdly to the lysosomes, due to the presence of mature saposin C. The defective saposin B. however, is rapidly degraded in the lysosomes. In this case, there is also a disruption of the regularity of the cysteine residues, suggesting that this could also alter the stability of saposin B (Henseler et al., 1996). These four mutations result in a deficiency of saposin B, and consequently in a metachromatic leukodystrophy-like phenotype. The discovery of this variant form of metachromatic leukodystrophy emphasizes the importance of saposin B in the degradation of glycosphingolipids. A deficiency in saposin C has been reported to cause a variant fon of Gaucher disease (Christomanou et al., 1986). Two point mutations in the saposin C coding region of the prosaposin gene have been reported. Schnabel et al. (1991) demonstrated that a G to T transversion results in a cysteine to phenylaline substitution in saposin C. The missinç cysteine residue may affect the tertiary structure of the mutant protein, leading to instability. A patient with this mutation had normal glucosylceramide B-glucosidase activity and yet an accumulation of glucocerebroside in the spleen and other tissues (O'Brien and Kishimoto, 199 1). The second point mutation in this patient was a cysteine to glycine substitution due to a G to T transversion (Rafi et al., 1993). Both mutations allow prosaposin to be synthesized and processed normally, but saposin C is rapidly degraded when cleaved from its precursor (Christomanou et al., 1986). A mutation resulting in total prosaposin deficiency has also been repotted by Harzer et al. (1989). Analysis of the tissues of two siblings having this disorder showed an accumulation of different sphingolipids such as glucosylceramide, galactosylceramide, and ceramide. Sequence analysis of the mRNA showed an A-T transversion in the initiation codon preventing prosaposin synthesis. Through these deficiencies and mutations, it has become clear how important prosaposin and its mature saposin proteins are in the degradation of glycosphingolipids. Without these proteins, lysosomal storage diseases occur, which are often associated with neurological deterioration, delajred nerve conduction time, accumulation of lipids in tissues, and other senous syrnptoms (Henseler et al., 1996). These diseases have helped elucidate the physiological functions of saposin B and C, but more work must be done in order to clarifi the functions of the other two saposins (A and D). In nomal tissues, saposin D is the most abundant of the four activators derived from prosaposin (O'Brien and Kishimoto, 1991). However, massive accumulation of saposins (particularly saposin A) are found in the brain of patients with Tay-Sachs and Sandhoff disease (O'Brien and Kishimoto, 1991). The question anses as to why increased levels of saposins are found in storage diseases where saposins have no known role in the degradation of the accurnulated material. ln Tay-Sachs disease for example, GM2 ganglioside accumulates in the lysosomes due to a deficiency of hexosaminidase, yet enonnous quantities of saposin A also accumulate in these organelles (O'Brien and Kishimoto, 1991). In liver fucosidosis, Saposin A and D also accumulate in the lysosomes (O'Brien and Kishimoto, 199 1) Thus, studies need to be camed out to detemine whether or not the saposin accumulation plays a significant role in these diseases. F) Altemntive Splking of Prosnposin niRNA Alternative splicing ofthe prosaposin mRNA in normal humans leads to the inclusion or exclusion of exon 8, a 9 base pair sequence in the saposin B domain, coding for Gln-Asp- Gln (Diagram 3). The alternative splicing can lead to total inclusion of exon 8, total exclusion of exon 8 or a partial inclusion of exon 8, consisting of the segment encoding the amino acids Asp-Gln (Diagram 3) (Holtschmidt et al., 1991, Lamontagne and Potier, 1994, Henseler et al.. 1996). A splice site with low fiequency has been located in the second intron of the saposin B region of prosaposin (Diagram 1) (Roman et al., 1991 ). Although exon 8 is commonly found in normal individuals, it has also been detected in association with certain disorders. Zhang et al. (1990) found a 33 base pair insertion which included the 9 base pair exon 8, in a patient who was diagiosed with a form of metachromatic leukodystrophy. The investigators examined the sequence of the 33 bp insertion of their patient in an attempt to understand the mechanism controlling the alternative splicing of the prosaposin gene in normal individuals. They observed that the two nucleotides upstream from the insertion consisted of AG. which is suspicious since these same two nucleotides also proceed the 9 base pair, and the 6 base pair insertions of exon 8. Thus, they proposed that the presence of an AG upstrearr fiom exon 8 acts as the signal for the alternative splicing of this exon in normal individuals. In the patient with the 33 base pair insertion, a point mutation must have generated a new acceptor splice site (AG), which caused the insertion of 33 base pairs of intronic RNA into the mature transcript (Zhang et al., 1990). Although the alternative splicing of the prosaposin mRNA has been shown to occur in normal individuals, and has recently been studied in more detail, the significance of this process is still poorly understood. Rat SGP- 1 CAGATGATGATGCACATG CAACCCAAG Normal human CAG ATG ATG ATG CAC ATG CAA CCC AAG Normal human + 9 bp CAG ATG ATG ATG CAC ATG 4 CAA CCC AAG CAG GAT CAG
Patient + 33 bp CAG ATG ATG ATG CAC ATG 4 CAA CCC GAG ATT TCC TGT TTT TTT GTT CAA CAG CAG GAT CAG
Schematic diagram: Nucleotide sequences showing complete homology on either side of the insertion site between rat SGP-1 (Collard et al., 1988), normal human (Nakano et al., 1989), normal hurnan with 9 bp insertion, and patient with 33 bp insertion (Zhang et al., 1990). Bold nucleotides represent the 9 bp of exon 8 (Adapted from Zhang et al., IWO).
The alternative splicing of the prosaposin gene is thought to be tissue specific according to Lamontasne and Potier (1994). They have found that the form of prosaposin mRNA containing the 9 bp insertion of exon 8 is synthesized in fibroblasts, brain, and pituitary gland, whereas the mRNA devoid of the exon 8 is present mostly in liver and lymphoblasts. Although no other researchers have examined the presence or absence of exon 8 in diflerent tissues or ceIl cultures, this study has led the investigators to conclude that the alternative splicing of exon 8 is most likely occurring in a tissue specific manner. Lamontagne and Potier (1994) have also demonstrated that a synthetic peptide for saposin B, which contained the 3-amino acid insertion encoded by exon 8, does not bind CM, ganglioside and increases the peptide's binding affinity for sulfatide and sphingomyelin. Thus, they suggest that the alternative splicing of prosaposin mRNA may give saposin B differing binding specificities according to the sphingolipid contents in certain tissues (Lamontagne and Potier, 1994). Henseler et al. (1996), have perfoned similar expenments to examine the possible biological function of the 9 or 6 base pair insert of exon 8. They developed three recombinant prosaposin proteins, with or without the 2 or 3 amino acid insertion respectively, and studied the ability of each prosaposin isoform to stimulate the degradation of sphingolipids. Henseler et al. (1996) have concluded that al1 three forms are capable of stimulating the degradation of sulfatide and globotriasylceremide in cultured fibroblasts to the same extent. In addition, their results indicate that the three polypeptides encoded by the different prosaposin cDNAs are not processed differently. The three isofoms of saposin B were also found to be stable and function in similar ways (Henseler et al., 1996). Furthemore, these investigators report that the 3-amino acid insertion may be located between the third and fourth helix of the proposed mode1 for the saposins, desctibed by Stevens et al. ( 1993). This would indicate that these three extra amino acids could affect the binding affinity of lipids in this region, which supports the findings of Lamontagne and Potier (1 994). and suggests a possible role for this exon. Clearly, further research is needed to be done on the structure of saposin B, and its different isoforms in order to understand their fùnctional significance.
3. Saposin-Like Proteins (SAPLIPs) The polypeptide motif characterized by the location of six cysteines and several hydrophobic residues is not only found in saposins A-D, but is also located in several other proteins such as; acid sphingomyelinase (ASM), acyloxyacyl hydrolase (AOAH), surfactant protein B (SPB), and Entamoeba histolytica poreforming peptides (amoebapores) (Munford et al., 1995). Acid sphingomyelinase (ASM) is a lysosomal enzyme that hydrolyses sphingomyelin to ceramide and phosphocholine. A deficiency of this protein results in a lysosomal sphingomyelin storage disorder (Munford et al., 1995). Acyloxyacyl hydrolase (AOAH) is a lipase found in phagocytic cells that cleaves fatty acyl chains from bacterial lipopolysaccharides (LPSs) and glycerolipids. LPSs can be compared to eukaryotic glycosphingolipids (Munford et al., 1995). Surfactant protein B (SPB) is a hydrophobic protein produced by alveolar type II cells, and it helps to increase the rate of spread of surfactant along the water-air interface in the alveoli of the lung. The disulfide linkages in SPB are identical to those in saposin B. SPB is soluble in organic solvents and it binds to phosphatidyl glycerol which is a lipid present in surfactant (Munford et al., 1995). Amoebapores are the pore forming proteins produced by the protozoan Entamoeba histolytica. These proteins may account for the ability of this parasite to destroy human cells. The amoebapores have hydrophobic and hydrophilic regions within their peptide sequence that resembles that of saposin C. In the region between two cysteine residues, Gys4 and CysS, the arnino acid sequences of the amoebapores and saposin C are quite similar, suggesting that this domain, in the third alpha helix, may contribute to the membrane-disrupting ability of these proteins (Munford et al., 2995). Munford et al. (1995) concluded that al1 of these saposin-like proteins share a motif that is formed by four amphipathic alpha helices (as in the proposed structure of saposin B), and that this structure may be interacting with lipids in different ways. For example, this core structure may fûnction in allowing the protein to act as an enzyme cofactor, as the saposins do. It could allow the protein to act as a transporter for lipids between and within membranes, the way that prosaposin and saposins are thought to act. This motif could allow the proteins to interact with phospholipid bilayers as in the case of saposins, SPB, and arnoebapores. It could help the proteins carry out enzyrnatic reactions as in ASM and AOAH, or fûnction in reducing surface tension at the lipid-air interface for the SPB protein. Finally, this core structure could allow fo: ixitracellular protein targeting in prosaposin and AOAH, and protein-protein interactions in saposins and SPB. Thus, the SAPLIP motif is comparable to other multi fùnctional protein domains which are structurally similar but capable of fùnctioning in many different ways (Munford et al., 1995).
4. General Mechanism of Alternative Splicing A) RNA Synthesis Mature mRNA in eukaryotes is produced by the stepwise action of many protein and RNA molecules. RNA synthesis (also called DNA transcription) is a very selective process, hence only part of the DNA sequence is transcribed to produce nuclear RNAs, and only a small part of the nucleotide sequences in the nuclear RNAs continues to be processed to become RNA in the cytoplasm (Young, 1991 ). Three RNA polymerases exist and function in making RNA in eukaryotes. RNA polymerase 11 transcribes the genes whose RNAs are translated into proteins, while polyrnerases 1and Il1 make large RNAs and small RNAs (tRNA), respectively. The average length of a cornplete RNA molecule produced by RNA polymerase 11 is around 7000 nucleotides, which is much longer than a 1200 nucleotide RNA molecule needed to code for an average size protein of 400 amino acids (Young, 1991). This indicates the complex structure of eukaryotic genes which contain long intron sequences that are later removed fiom the RNA (Sentenac, 1985). The fieshly synthesized RNA molecules in the nucleus are called primary transcripts. These primary transcripts contain a capped 5' end which is formed by the addition of a methylated G nucleotide, which functions in the initiation of protein synthesis, and protects the growing RNA transcript from degradation. The 3' end of the primary RNA transcript is cleaved at a specific site and a poly-A tail is added to the cut end. This poly-A tail aids in the export of the mature rnRNA fiom the nucleus, serves as a recognition signal for the ribosome, and appears to affect the stability of some of the rnRNAs in the cytoplasm (Proudfoot, 1989). Most of the RNA in these primary transcripts is unstable and will be eventually degraded, thus the mRNAs produced fiom these transcripts only consist of a small part of the total RNA of the cell. The primary transcript can therefore be considered as a copy of a gene containing both exon and intron sequences, and these intron sequences are cut out of the transcript so that the resulting mRNA molecule directly codes for a protein (Hickey et al., 1989). After the intron sequences are cut out, the exon sequences on either side are joined. This is referred to as RNA splicing which occurs in the nucleus (Hickey et al., 1989).
B) RNA Splicing RNA splicing is a complex mechanism which involves many different proteins and RNA molecules. Small nuclear ribonucleoproteins, or snRNPs are small RNAs which have been located in the nucleus and are believed to recognize specific RNA sequences located at the junctions between intron and exon sequences. These particles together with various proteins pair up and form a complex called the spliceosome, that catalyses RNA splicing (Wu and Maniatis, 1993). The only conserved sequences in introns are those required for intron removal, and are found at the ends of an intron. At the 5' end of an intron, there is a conserved sequence which is called a GT (GU) donor splice site, and at the 3' end is an AG acceptor splice site. These sequences are necessary to define a splice junction (Balvay et al., 1993). The RNA splicing pathway has been demonstrated in vitro and begins with the snRNPs (Ul, U2, US, U4N6) assembling with specific proteins to fonn a large ribonucleoprotein complex called the spliceosome. Following this assembly, the nucleotide A fi-om the 3' AG acceptor splice site attacks the 5' splice site and cleaves it. This cut 5' end of the intron then becomes linked to the A nucleotide and forms a lariat (loop-like structure). Finally, the resulting 3' hanging end of the first exon sequence adds to the beginning of the second exon sequence, cleaving the RNA molecule at the 3' splice site. This results in the two exons being joined while the intron sequence is released, in the fonn of a lariat, and is degraded in the nucleus (Balvay et al., 1993, Wu and Maniatis, 1 993).
C) Alternative RNA Spiking Usually the RNA processing machinery ensures that each 5' splice site pairs only with the 3' splice site that is closest to it downstream (5' to 3'). However, the simple 5' to 3' splicing can be altered by certain mechanisms which allow a single gene to produce different rnRNAs and thus, several different proteins (Goguel et al., 1991). For example the inactivation of a splice site may occur by a single nucleotide change which would cause the formation of a new cryptic site nearby. In addition, any other single nucleotide changes could create new splice sites by changing the sequence of an intron or exon (Holtschmidt et al., 1991). Thus, through this process, the primary RNA transcript may be spliced out in different ways, producing different polypeptide chains for the same gene (Diagram 3). This mechanism is referred to as alternative RNA splicing, and occurs in higher eukaryotic cells indicatins the evolution of the genes in these organisms (Rio, 1992). Constitutive alternative splicing occurs when the splicing mechanism is unable to distinguish between two or more alternative pairings of 5' and 3' splice sites. As a result these different choices are made at different times and several versions of the protein are produced in al1 the cells where the gene is expressed (Green, 1991). Alternative RNA splicing can also occur in a regulative rnanner, where there is a switch from the production of a non-fiinctional protein to the production ofa fuiictional one. Regulative alternative splicing can also generate different versions of a protein in different ce11 types according to the needs of the ce11 (Bickmore et al., 1992, Green, 1991). This second function seems to be the reason for the alternative splicing of the prosaposin gene (Diagram 3), where exon 8 is either present or absent in the prosaposin mRNA of different tissues (Lamontagne and Potier, 1994).
5. Objective of the Present Research Until now, the examination of the alternative splicing of prosaposin mRNA has only been performed on a few tissues and ce11 cultures by Lamontagne and Potier (1994). In addition, these studies were only done on human cells. Recently, our laboratory cloned the mouse SGP-1 (prosaposin) gene (see NCBI Genebank accession number U57999), and thus, it was possible to study the structure of this gene in more detail . Furthemore, the moiise is a useful species for experiments that can not be peFformed on humans, such as those experiments involving genetic manipulations. The objective of the present research is to perform a comprehensive study of the 1) alternative splicing of prosaposin mRNA using RT- PCR and specific primers; 2) mRNA expression of prosaposin using Northern Blot analysis; and 3) the tissue distribution of its translated product in several tissues of the mouse, using light microscopie immunocytochemistry. These experiments will demonstrate for the first time, the tissue specific alternative splicing of SGP-1 mRNA and the distribution of its translated product in eight different tissues of the mouse. III. DIAGRAMS Diagram 1 : Structure of SGP-llprosaposin with its saposin domains Diagram 1 Diagram illustrating the abbreviated structure of SGP-l/prosaposin with saposin domains A, B, C, D (Gray boxes), signal peptide (Black box), glycosylation site (Y), and an alternative splice site in the saposin 8 region (arrow). The lines located in between each saposin domain indicate protein sequences that are removed during post- translational proteolytic processing (adapted from Kishimoto et al., 1992). Diagram 2: The saposins have conserved positions of their amino acid sequences Diagram 2 Diagram illustrating the abbreviated structure of SGP-l/prosaposin with saposin domains A, B, C and D (Gray boxes), and a signal peptide (Black box). The identical placement and alignment of the cysteine and asparagine residues in each saposin domain is demonstrated by the row of letters situated above each gray box. This indicates the areas within the saposin proteins, where the positions of the arnino acids are conserved. Cysteine residue; C, asparagine residue; N. Diagram 3: Alternative splicing of exon 8 Diagram 3 Schematic representation of three of the four exons that code for saposin B (exon 6 is not shown). Exon 7 and exon 9 (Gray boxes) are always present in the mature mRNA transcript for saposin B, whereas exon 8 (white box) is either spliced into the mRNA transcript (illustrated above the diagram), or it is spliced out with the introns, as indicated below the diagram. The lines joinning the boxes represent the intronic sequences that are removed from the mRNA transcript. IV. Materials and Methods
1. Total RNA Extraction A) Repwation of Solutions This technique required al1 solutions and equipment to be treated with DEPC (diethylpyrocarbonate, 50 pl 1250 ml), in order to prevent the degradation of RNA. RNA is very sensitive to RN- and therefore @oveswere wom during the course of the experiments. Some solutions needed to be prepared prior to the day of RNA extraction and are described below. The lysis buffer was made by dissolving 59 g of Guanidine monothiocyanate in 83 ml DEPC 40at 50°C,adding 10 mM EDTA, 50 mM Tris HCI pH 7.5, and just before use, 8% beta mercaptoethanol (O-ME) was added. A 4 M lithium chloride solution was prepared by dissolving 16.96 g of lithium chloride in 100 ml DEPC H,O. The homogenization buffer consisted of 12 ml of a 5 M NaCl solution, 0.5% SDS, 5 rnM EDTA, and 20 ml of a 1 M Tris HCI solution (pH 7.5).
B) Tutd RNA Isolaîion and Pirn!cntion Adult male CD4 mice purchased fiom Charles River Canada Inc. were anaesthetized with sodium pentobarbital(0.1 dl00g body weight), and eight different tissues (muscle, testis, heart, lung brain, pancreas, spleen, and kidney) were isolated. The tissues (1 g) were placed in 7 ml of iysis buffer and 570 pl of U-MEin separate corex tubes. They were then homogenized with a Polytron homogenizer, and 7 ml of chloroform was added to each tube. The sarnples were centrifbged at 5 K for 7 minutes in a rotor centrifuge, and the supernatant was carefully removed and placed into a new tube. Lithium chloride (7 volumes) was then added to each tissue homogenate, and the tubes were lefi on ice for 15-20 hours in the fiidge. The following day, the sarnples were centrifùged at 1 1 K for 30 minutes after balancing tubes with lithium chloride. The pellets were then resuspended in 15 ml of LiCI, and the sarnples were centrifugeci again for 30 minutes at 4°C. This step eliminated any excess D-ME. The pellets were then resuspended in homogenization buffer ( 1-2 ml), treated with proteinase K (1 00 &ml) and lefi at 45 " C for 30-45 minutes. Equal volume (2-3 rd) of chioroform was added to the samples, and they were centrifùged at 5 K for 7 minutes. The supematants were subsequently removed with a disposable pipette, and placed in new tubes containing another 2-3 ml of chlorofonn. These solutions were centrifùged at 5 K for 7 minuta, and the supematants were removed, placed in new tubes containhg phenoVchlorofoim (2-3 ml), and centrifùged at 5 K for 7 minutes. The top layers were then carefùlly removed and transferred to fkesh tubes. Then, 2.5 volumes of ethanol was added to the sarnples, and they were kept at -70°C for 24 hours. This step allowed the RNA to precipitate. The tubes were centrifùged for 20 minutes at 1 1 K the next day, and the pellets were resuspended with 300 pl DEPC water and transferred to sterile eppendorf tubes. 9 pl of a 5 M NaCl solution and 700 pl of ethanol were added to the sarnples, and they were placed at -70" C for 1 hour, and then centrifùged. The supematants were poured out and the pellet was dissolveci in DEPC-H20. The RNA was measured using a spectrophotometer. The total RNA was stored at -70°C to avoid any degradation.
2. Anaiysis of Total RNA Using the Polymerase Chain Reaction A) Designing PCR Prlrnen Three primers narned Primer 1, Primer 2, Primer 3 (Table l), were designed using an Oligonucleotide Software Primer Program and were sent for synthesis at the Sheldon Biotechnoiogy Centre at Mcgill University. These primers were designed to anneal to a specific area of the cDNA encoding for the SGP-I protein of the mouse, recently cloned in our laboratory. The area of interest within the SGP- 1 cDNA sequence is located within the saposin B dornain of the gene, which may contain the alternatively spliced exon 8 being studied. The first set of prirners (Primer 1 and Primer 2) were both 2 1 nucleotides in length, and were venfied with the computer program to have; an appropriate GIC content of 50%, no significant complementarity to one another, similar melting temperatures, and good secondary structures containing a free 3' end. Both prirners were diluted to a final working concentration of 20 PM. The location of these primers was chosen to yield two possible PCR productq depending on the tissue examined; either a 115 base pair fiagrnent containing the exon 8, or a 106 base pair fiagrnent devoid of the exon 8. These primers (Primer 1 and Primer 2) were used to show the tissue specific alternative splicing of the SGP- 1 gene in the eight diflerent tissues of the mouse, using Reverse Transcription- Polymerase Chain Reaction (RT-PCR). The second set of pnniers (Primer 1 and Primer 3), differing fiom the first set, in the length of Primer 3 (20 nucleotides) and in the distance between the two primers, containecl an average G/C content of 55%, were not complimentaiy to one another, had similar melting temperatures, and good secondary stiuctures. These two primers were used to perfonn RT-PCR on only two tissues (brain and kidney), and appeared to produce stronger and more abundant PCR products than the previous set of primers. Therefore, these PCR products were subsequently used for subcloning and sequencing. These two primers were expected to produce two possible PCR products; a 146 base pair fragment representing the tissue devoid of exon 8 (kidney), and a 155 base pair fragment containing exon 8 (brain).
Table 1
GC Content 57% ACAGAAGCCAGCCA primer GCACACA Primer 2- 2 1 nucleotides 45% CGTGTCTGACATATG antisense primer CAAGAA Primer 3- 20 nucleotides 55% TGGACCACGTGAAGG antisense primer AGGAT
R) RT-KR Following total RNA extraction, 5 pg of RNA fiom the eight different tissues of the mouse, were used as templates for cDNA synthesis using reverse transcription. Fist, the mixture of total RNA and DEPC-H,O (final volume 6 pl) was denatured by heating at 70 OCfor 10 minutes. The mixture was then chilled on ice. To this mixture was added; 1 pl of deoxynucleotide triphosphates (dNTP: 0.1-0.2 mM final concentration of dATP, dCTP, dGTP, dTTP), 5 pl of 5X AMV RTase buffer (Promega), 40 pmol of an oligo dT primer, 20 units of RNasin, 5-7 units of AMV reverse transcriptase (RTase 10 unitdpl), and DEPC-HO to a final volume of 20 FI. This reaction mixture was incubated at 23°C for 10 minutes, at 42°C for 45 minutes, and then heated to 95°C for 5 minutes. The tubes were kept at 4OC until PCR was ready to be perforrned.
C) Polymerase Chuin Redon (PCR) 5 pl of the single stranded cDNA item the previous step was used as a template for PCR. To this template was added, 1 pl of dNTP (Mconcentration of0.1-0.2 mM), IO pl of 10X PCR buffer (final concentration of 1X), 1 pl of Primer 1 and Primer 2 or Primer 3 (final concentration of 0.1-0.2 PM), ddH20(double distilled sterile water), and 0.5-0.75 pl of Expand Long Ternplate PCR System enzyme (Boehringer Mannheim) or Taq DNA Polyrnerase (final concentration 1-5 unitdl00 pl Phamacia Biotech). The reaction mixture was brought to a final volume of 100 pl. The PCR sarnples were then placed in an automated Programmable Thermal Controller (PTC- 100, MJ Research Inc.), and to the top of the mixture, an overlay of 30-60 pl minera1 oil was added to prevent evaporation of the solution. Different temperature cycles were used in order to ampli$ the cDNA. When using the expander enzyme, the PCR mixture demibed above was denatured for 2 minutes at 94°C. If Taq DNA polymemse was being used, the samples were placed in the PCR machine devoid ofthe Taq enzyme, demtured for 5 minutes at 94"C, the temperature was then lowered to 80°C and the Taq enzyme was added to the PCR samples. Taq DNA polymerase is more stable at this lower ternperature. Following this initial denaturing step, the PCR samples were denatured for another 30-60 seconds at 94°C. The primers annealed to the cDNA for 30-60 seconds at the primer annealing temperature, which was previously deterrnined, and the primers were subsequently extended for 45-60 seconds at 72°C. These four steps were then repeated 20-30 more times for amplification, and lastly there was a final extension for 7-1 5 minutes at 72"C, which enabled the Taq DNA polymerase or the Expander enzyme to add on the poly A tail. The resulting PCR products were then kept at 4°C. Ail these steps may be varieci depending on the primers used and the type of DNA being amplified. Before performing any additional rounds of PCR to further arnplify the desired product, it was recomrnended to optimize the PCR conditions in order to know what results in the rnost specific and abundant PCR product. Optimizing PCR conditions involved changing annealhg temperatures, increasing or decreasing the arnount of cycles, varying the concentration of DNA template, and changing the amount of enzyme added. Once the proper conditions had been attained, PCR was able to be repeated again using the previous PCR product as a template. This further arnplified the desired DNA segment so that a nice arnount was visible on a gel electrophoresis, and thus, the DNA could be easily used to subclone into a vector and to be subsequently sequenced.
D) Andysis of KRProducts (7) Agirose gel electrophores~s A 1-2% agarose gel was prepared by dissolving 0.5-1 g of low electroendosmosis agarose in 50 ml TAE buffer (final concentration I X), and 2.5 pl of ethidium bromide was added (0.5 ,ug/ml) to this solution. The agarose in TAE buffer was heated and then poured into a container containing a comb, and was left to harden. The PCR products (10 pl-1 00 pl) were prepared by adding to them a 6X loadimg buffer (0.25% bromo phenol, 0.25 % xyiene cyanol, and 30% glycerol at a final concentration of lx). These sarnples were subsequently loaded into separate weUs of the agarose gel. A standard 100 base pair ladder (Phannacia Biotech) was used as a marker for estirnating the size of the PCR products. The container was filled with 1X TAE buffer, and the gel was run for 1-3 hours at 35-65 volts. The PCR products (DNA bands) were then visualized under UV illumination and photographed (Polaroid-57 film). The corresponding size of the DNA fiagments was determined by comparing them to the DNA marker.
(Nl Non Lk,m/wing Po&ctcry/nmide Gel Eiec trophoresis This technique allowed for the separation of small DNA fiagments and thus could resolve the 9 base pair difference between some of our PCR products. First, the apparatus was assembled, glass plates were put together with spacers in between them to cast the gel. To prepare the gel solution, 12 ml of a 30% acrylamide solution, 14.4 ml dd H20,630pl of a 10% ammonium persulfate solution, 3 ml of 10X TBE buffer, and 15 pl of TEMED (N,N,N',N'- tetramethylenediamine), were rnixed together. This solution gave a 12% gel which was recornrnended for the separation of 50-200 base pair fiagments, corresponding to Our fiament Si.The gel solution was then loaded between the glas plates, a comb was inserted, and it was lefl to polyrnerize for a minimum of 1 hour. Once the gel was klly polyrnerized, the PCR samples of interest were rnixed with a 6X loading buffer, that was diiuted in 1X TBE,to a final volume of 20-35 pl. These samples were loaded ont0 the gel and the gel was nin in the fiidge overnight at 30-60 volts. The PCR products were visualized under W illumination and photographed.
E) Isulaîiun of the DNA Fmgments Amplped by KR In using the RT-PCR method descnbed above with Primer 1 and Primer 3 (Table 2), two PCR products of 146 (kidney) and 155 (brain) base pairs were produced, run on an agarose gel electrophoresis, and cut fiom the 1% agarose gel. These DNA fragments were then purified using the Agarose Gel DNA Extraction Kit from Boehringer Mannheim. This kit consisted of a Sica ma* nucleic acid binding and washing buffers. It fùnctioned in separating the DNA fiom the other constituents, by having the DNA bind to the matnx in the presence of chaotropic salts. This separated the DNA fiorn the impurities that may have interfered with subcloning and sequencing methods that followed. The eluted DNA diluted in dM20, was subsquently measured on a DU-64 Beckrnan spectrophotometer.
3. Cloning of the PCR Products A) Ligntion of the KRproduet into a pGEW- T Vector In order to obtain optimal ligation. a 1: 1 molar ratio of the pGEM1'T Vector to the PCR product was used (in a 10 pl final reaction mixture). The pGEM*)T Vector is about 3 kilobases in length, it has been linearized at base 5 1 of the circular pGEM%Zf(+) Vector fiom which the pGEMW-TVector is denved, and a T overhang has been added to both 3' ends (Fig. 3). The pGEW-T Vector is supplied at a concentration of 50 ng/pl, from Promega. The ligation reaction consisted of 1 pl of T4 ligase 10X bufEer, 1 pl of the pGEhrfiT Vector, 1 pl of T4 DNA Ligase, PCR product ( for a final ratio of 1 :1 for pGEMO'T vector:PCR product), and ddH20 to a final volume of 10 pl. This mixture was incubated at 15 OCovemight . The ligase was then inactivated in a thermal cycler the next day, and 3 pl was removed from this reaction for the transformation step that follows.
B) Trnnformaf!*oon of Ligated PCR:pCEM@-TVector hto High Eflciency Comptent Cells Transformation was perfomed using JM 109 High Efficiency Competent Cells (>1 X 108cfÙ/ pg DNA homega). LB/arnp/IPTG/X-Ga1 plates were prepared by autoclaving 1 1 g of NZCYM Broth from GIBCO BRL,and 7.5 g of agar in 500 ml of W,O. Then, 300 pl of X-Gd, 250 pl of I M IPTG and 1 ml of ampicillin (50 pgl ml) were added to the rnixhire et 45-50°C, and poured ont0 petri dishes. The ligated PCR product:pG~M@-TVector reaction mixture was centnfùged, and 3 pl of this was added to 50 pl of High Efficiency Competent Cells (JM 109 E.Coli bacteria). These cells are extremely fiagile and were handled carefùlly. The mixture was kept on ice for 20-30 minutes, and then the reaction was heat shocked for 1 minute at 42°C exactly. The sarnples were put back on ice for 2 minutes, and 200-400 pl of S.0.C media was then added to this 50 pl mixture, mixed gently, and incubated on a shaker for 1 hour at 37" C. The transformation culture was then spread ont0 2-3 separate LB/TPTG/X-GaVampicillin plates, inverted, and incubated overnight at 37°C. The following day, the amount of colonies present on the plates was compared to the control plate to ensure that plating results were satisfactory. White colonies were expected to contain inserts.
Q Idenhifi~~~onof the Positive Clones In order to determine which colonies actually contained the insert of interest, PCR was performed on 6 white colonies of both brain and kidney samples. This saved time in having to grow al1 12 colonies and extract ail the plasmid DNA fiom each sarnple. Thus, each white colony was picked up by a pipette tip and piaced in a mixture containing; 2.5 pl of 10X PCR buffer, 0.25 pl of eadi nucleotide, 0.25 pl of T7 and SP6 pnmers, and stede water for a final volume of 20 pl. The bacterial colony was rnixed well with the solution, and then denatured for 5 minutes at 94'C. The temperature was subsquently decreased to 80°C, and 5 ,ul of diluted Taq DNA polymerase was added. PCR was then performed on the bacterial colonies, using the same protocol for PCR described above. Al1 twelve colonies were also re-incubated at 37°C on a fiesh LB/IPTG/ X- GAV ampicillin plate for 3-4 hours in order to use them in later expenments. A 1% agarose gel (described above) was run on the 12 PCR products in order to determine which colonies were positive, and contained the insert of 146- 155 base pairs. Based on these PCR results, two of the positive colonies (one fiom brain and the other fi-om kidney) were chosen to be sequenced.
D) Isolaîion of Recombinant PlddDNA These two positive white colonies (confirmed by PCR), that were previously incubated on a fresh LB plate, were picked up and amplified by growing them in 50 ml of LB broth, containhg 100 pl of ampicillin, and were incubated ovemight on a shaker at 37°C. The arnplified recombinant DNA was then extracteci fiom the bacterial DNA, using the Plasmid Mini Kit from QIAGEN, Montreal.
4. Sequencing of the PCR Products A) Phpuhg Plmmid DNA for PriwAnnealing The punfied piasmid DNA samples from kidney and brain were denatured by taking 8 pl of the template DNA and mixing them with 2 pl of 2 M NaOH. These samples were then centrifùged briefly and incubated for 10 minutes at roorn temperature. Following this incubation, 3 pl of 3 M sodium acetate, 7 pl distilled water, and 60 pl of 100% ethanol were added to the solutions and rnixed well. They were subsequently put on dry ice for 15 minutes, and the precipitated DNA was collected by centrifùging the samples for 10 minutes. The resulting supematents were removed and the pellets were washed with ice cold 70% ethanol. The solutions were centrihged again for 10 minutes, the supernatants discarded, and the pellets were dried and redissolved in distitled water. For the annealing reaction, 10 pl of the denahired template DNA fiom above (1-2 pg of DNA), 2 pl of T7 or SP6 primers, and 2 pl of anneding buffer were mixed together. The tubes were vortexed gently and the solutions were incubated for 20 minutes at 37°C. The samples tiom kidney and brain were sequenced from both ends of the plasmid DNA using the T7 and SP6 primers (in separate reaction tubes), to ensure that the insert within the vector was sequenced.
B) Sequencing Reactions To the tubes containing the annealed templates and T7 or SP6 primers (for kidney and brain) was added, 6 pl of Enzyme Premix which consisted of 3 pl of labelling rnix-dATP or - dCTP (which are labelled with [c+~'S]) for each template DNA, 2 ,d of diluted T7 or SP6 DNA polymerase (diluted with enzyme dilution buffer 1.5 unitdpl), and 1 pl of labelled dNTP (1 pl=l O @Ci). These components were rnixed gently, centrifiiged briefly, and incubated for 5 minutes at room temperature. in the mean time, the four sequencing mixes were wmed at 37°C for at least 1 minute. These sequencing mixes consisted of separate tubes containing 2.5 pl of an A Mix, C Mix. G Mix, and T Mix (Sequencingm Kit). Mer the 5 minute incubation of the Labelling Reaction above, 4.5 pl of this reaction was transferred to each of the four pre-warmed sequencing mixes. These reaction mixtures were incubated for 5 minutes at 37T, and then a Stop Solution was added to each tube and mixed gently. The samples were finally heated to 75-80'C for 2 minutes and loaded into the wells of the sequencing gel (described below). AI1 solutions and components needed for these reactions were provided by nSequencing" Kit fiom Pharmacia LKB.
CJ Sequencing Gel The gel solution was prepared by adding 41 g of urea, 6 ç of aciylamide, 0.27 g bis- aciylamide, 10 ml 10X TBE buffer, and 44 ml H,O. To 70 ml of this gel solution, 150 pl 20% Ammonium Persulphate (APS), and 60 pl of TEMED were added. The gel was poured in between clean, dry assembleci glass plates, and thin spacers and the straight side of the sequencing comb was placed in between the plates. The clamps were then put at both top sides of glass plates, and the gel was lefi to polymerize (1 hour). The gel was then placed in the electrophoresis apparatus, buffer was added, the comb was removed, and the wells were washed with TBE buffer. The gel was pre-iun for 1 hour at 50 watts, or until plates were hot. The samples were denatured for 3-5 minutes at 90°C before loading. The sequencing comb was then placed in between the plates, with its teeth barely touching the gel. Then 3 pl of each sequencing sample (four samples for brain and kidney with either T7 or SP6 primers) were loaded into separate wells of the gel. The gel was run for 90 minutes at 50 watts. Once the running of the gel was completed, the tape around the gel was removed and the plates were separateci. The bottom plate with the gel was imrnersed into a fixative (10% methanol and 1W acetic acid solution) for 20 minutes, and then kimwipes were used to remove the excess fixative f?om the surfice of the gel. A 3 mm wattman paper was placed on top of the gel and the gel was subsequently dried for 30-60 minutes. The gel was then exposed on an X-ray film for 24 hours and the film was developed.
5. Analysis of RNA using Northern Blot Hybridization
A) Agurose~Formddehydè(;el Electrophoresis To prepare the gel solution, 1.2 g of agarose was dissolved in 10 ml of 10X Hepes-EDTA (200 rnM Hepes and 10 mM EDTA, pH 7.8) and 73.8 ml autoclaved water. The solution was heated and stirred until it appeared clear, and it was then cooled to 60°C in a water bath. Then, 16.2 ml of 37% formaldehyde was added, and the solution was poured into gel container. Total RNA samples previously isolated fiom twelve different mouse tissues (see section 1 above) were prepared by mixing them with 100 pl 10X Hepes-EDTA, 500 pl formamide, and 160 pl formaldehyde to rnake a 4X sarnple buffer. 5 pl of total RNA (5-20 pg) was denatured by adding 15 pl of this sample buffer. The samples were wmed at 60°C for 5 minutes, and 5 pl of bromo phenol blue was added to each sarnple, and they were loaded ont0 the 1.2% gel contairing a running buffer that consisted of 100 ml 1OX Hepes EDTA and 162 ml of a 3 5% formaldehyde solution. The gel electrophoresis was nin for 24 hours at 35 mV.
R) Tramfer of RNA frum Gel to Membrane The gel was placed in a RNase-fixe glass dish and nnsed with several changes of distilled water. This step removed the fomaldehyde which would have reduced the retention of RNA by nylon membranes. The gel was then soaked in 20X SSC for 45 minutes. The transfer apparatus was set up and consisted of a vacuum blotting unit fiom Pharmacia LKB 2016 vacugene. The wet membrane (Hybond-N Membrane from Arnersham, Life Science) was placed on the wrface of the gel and mewas taken to prevent getting air bubbles under the membrane, The surFace of the membrane was covered with 400 ml of 20X SSC (87.6g NaCl and 88.2 g sodium citrate, pH 7.0). The transfer was carried out for 1 hour and the vacuum unit was set to 50 units of pressure. The gel was removed and diswded while the nylon was soaked in a water bath for 5- 10 minutes, and then was placed on a filter paper and left to dry for 30 minutes. Once dry, the RNA was cross linked ont0 the membrane with UV light.
C) fiepwaîion of the RNA Robe The cDNA coding for the SGP- 1 protein of the rat was cloned into the pGEM-T vector, transformed into high efficiency JM 109 E.Coli bacteria, arnplified and isolated as aiready described above. The DNA was then linearized by restriction digestion for 2 hours at 37°C immediately downstream of the cloned fiagrnent (1 pl restriction enzyme Not 1,s pl of buffer, 2 pg plasrnid DNA and ddH20to a final volume of50 pl). The RNA probe was then synthesiied by adding 4 pl of labelling buffer, 1.5 pl of nucleotide mk:rATP, rGTP, rTTP, rCTP (2.5 mM from Promega), 2 pl of BSA (1 mdrnl), 1 pl 200 mM DTT, 1 pl human placental ribonuclease inhibitor (Promega), 2 pg of the purified plasrnid DNA, 100-20 ,diof [a-)'~]UTP fiom Dupont, and ddH,O to a final volume of 20 pl. To this reaction mixture was added 5 units of SP6 or T7 RNA polymerase, incubated for 1 hour at 40°C for SP6 and at 37°C for T7. The specific activity of the resulting probe was 108cpm/pg. Acid precipitation was then done to remove the unincorporated nucleotides using a spin column. The RNA was resuspended in 10 ml of Andy's buffer that contained; 100 ml of formamide, 80 ml of a 1 M NaH,PO, (pH 7.2), 800 pl of 250 rnM EDTA and 10 g of SDS. The solution was dissolved at 45"C, and then 10 ml of dissolved BSA in H,O was added to it. D) Hybn'clizution Anaiysis Before hfiridization, the membrane canying the immobilized RNA was soaked in ddH,O. Pre-hybridization and hybridization were cded out in glass tubes in a rotating commercial hybridization oven (Robbins Scientific mode1 2000). The membrane was placed, RNA side up, in the hybridiition tube for pre-hybridization by incubating with 10 ml of hybridimtion buffer (1 00 ml formamide, 80 ml of 1 M NaH,PO,, 800 PI of 250 mM EDTA, 10 g SDS and 10 ml of 2% BSA) overnight at 60°C in the rotating hybridization oven. The membrane was then incubated ovemight at 60°C for hybridization with the RNA probe, dissolved in 10 ml of hybridization buffer describeci above. Following hybrid'ion, the membrane was washed three times in 2X SSCIO. 1% SDS for 5 minutes at room temperature. One more wash was performed using a prewarmed O.2X SSCIO. 1% SDS for 30 minutes at 68°C. These washes ensured that non-specific binding was elirninated. The membrane was placed into a radioautographic cassette which contained KODAK film, and it was stored at -70°C. The film was exposed to the membrane for 24 hours and was then developed.
6. Analysis of the Translated Product of the SGP-1 mRNA Using Light Microscopy lmmunocytochemistry A) Tissue FPepnration Adult male CD4 rnice that were purchased from Charles River Canada Inc. were anaesthetized with sodium pentobarbital (0.1 cc1100 g) and perhsed through the heart with Bouin's fixative. An incision was made fiom the diaphragm of the animal up to the level of the sternum, enabling access to the rib cage. The heart was lifted up hmthe thoracic cavity and a perfùsion needle was inserted into the left ventricle. Mer the blood was cleared fiom al1 vessels with a ringer solution, Bouin's fixative followed for 10 minutes. Eight different tissues (muscle, testis, heart, lung, brain, pancreas, spleen, and kidney) were then dissected from the mouse, and immened in Bouin's fixative for 24 hours. The tissues were then dehydrated in graded ethanol solutions and sent for embedding in paraffin, sectioning, and mounting to the Pathology Centre at the Montreal Children's Hospital. B) Imtnunocytochem*stryusing Irnmunoperox~*daseStaining The irnmunoperoxidase staining of eight tissue sections of the mouse was canied out according to Oko 1988. The anti SGP-1 antibody was kindly supplieci by Dr. Michael Griswold from the Department of Biochemistry at Washington State University. 5 Pm thick paraffin sections of adult mouse muscle, testis, heart, lung brain, pancreas, spleen, and kidney were deparaffinized in xylene and then hydrated in a series of 5 minute graded ethanol solutions, starting from 100% and moving to 50% ethanol. During hydration, residual picric acid was neutralized in 70% ethanol containing 1% lithium carbonate. In addition, endogenous peroxidase activity was abolished in 70% ethanol containing 1% hydrogen peroxide. Once hydrated, the tissues were washed for 5 minutes in distilled water containing 300 rnM of glycine in order to block fi-ee aldehyde groups. Pnor to immunolabeling, the non-specific binding sites were blocked for 15 minutes in a TBS (TrisBase Buffered Saline containing 0.1% bovine serum dburnin, pH 7.4) solution containing 10% goat serum. This was accomplished by placing 40 pl of the solution ont0 a coverslip and then overtuming the tissue face of the slide onto the drop, thus ensuring that the entire tissue was treated with minimal fluid (Oko, 1988). These coverslips were then removed by rinsing the slides with TBS, and allowing the coverslip to slide off the tissue. Sections were then incubated at 3 7OC with the primary antiserum, (anti- SGP- 1 antibody diluted 1 :100 in TBS) for 1.5 hours. Folowing piimw antibody incubation, the sections were washed 5 times for two minutes in TBS containing 0.1% Tween-20 in order to remove any unbound antibody. The sections were then incubated for 30 minutes at 37°C with a goat anti-rabbit secondary antibody conjugated to peroxidase, which was diluted 1: 100 in TBS. The sections were subsequently washed in TBS containing 0.1 % Tween-20 for 2 minutes 4 times. The final reaction product was achieved by irnmersing the sections for 10 minutes in 500 ml of TBS containing 0.03% hydrogen peroxide, 0.1M irnidazole, and 0.05% diarninobenzidiie tetrachloride @AB, Sigma Chernical Co., St. Louis, Missouri). The DAB was polymerized in the presence of the peroxidase moleailes and hydrogen peroxide to fom an insoluble brown polymer which was deposited at the site of the antigen-antibody complex (Oko, 1988). The slides were washed with distilleci water to remove any excess DAB, and counterstained with 0.1% methylene blue for 5-1 0 minutes. The tissues were then washed in tap water, foilowed by distilled water, and subsequently dehydrated by passing the slides through 4 graded ethanol solutions (50% to 100% ethanol, one minute ah). Following this dehydration step, the sections were placed in xylene for 5 minutes, cleaned, mounted with pemount mounting media, exarnined and photogaphed using a Car1 Zeiss light microscope.
CJ Imrnunocytochemical Controls A negative control was performed for every sectio~and consisted of a section of rat testis which was irnmunostained with nomal rabbit serum. A positive control was performed by staining the testis from the rat tissue, since it has been well documented to have a good reaction with the anti SGP- 1 antibody. V. Results
1. Identification and Analysis of the Tissue Specific Alternative Splicing of the SGP-1 mRNA in the Mouse A) Amplifwîion of Trvo nNI1 Fragments using KR Three primers (Primer 1, Primer 2, and Primer 3) were designed to match the flanking region of exon 8, located in the saposin B domain of the SGP-1 gene (Fig. 5). This exon 8 is nine base pairs long (CAG GAT CAG), and encodes for three amino acids (Gln-Asp-Gln). The first two primers (Primers 1 and 2), were designed to be 106-115 nucleotides apart, and were chosen so that the area of the SGP-I mRNA that contained exon 8 could be amplified using Reverse Transcription-Polyrnerase Chain Reaction (RT-PCR).There were two possible PCR products that were expected to result using this method: a 115 base pairs long fiagrnent containing the exon 8 of interest, and a 106 base pair long fiagment devoid of exon 8. RT-PCR was performed on the total RNA extracted fiom eight different tissues of the mouse (muscle, testis, heart, lung, pancreas, brain, spleen, and kidney). The resulting cDNA was then arnplified using PCR and analysed by agarose gel electrophoresis. Observations of the electrophoresed PCR products under UV illumination showed that five of the eight tissues (testis, lung, pancreas, spleen, and kidney) contained DNA fiagnents slightly srnaller than the bands of the three remaining tissues (muscle, heart, and brain). This was confimed when the sarne PCR produds were resolved on a 12% polyacrylarnide gel electrophoresis (Fig. 1). Cornparison of the band mobility relative to known standards, revealed that the larger fiagment was 1 15 bp and the srnaller one 106 bp. Another interesting observation in the polyacrylarnide çel was that the band correspondmg to muscle not only consisted of the larger 1 15 base pair fiagment, but also seemed to have a faint lower band of 1O6 base pairs (Fig. 1, lane 1). In order to confinn unequivocally that the DNA fiagrnents from brain, muscle, and heart contained the 9 bp insert ofexon 8, and that testis, lung, pancreas, spleen, and kidney did not, two tissues representing these groups (brain and kidney) were chosen for sequencing. To ensure that a good mount of DNA was attained for subcloning and sequencing, the presence of strong bands pnerated by the PCR products were verified by agarose gel electrophoresis. To further confirm the presence of different species of mRNA due to tissue specific alternative splicing, a second set of pnmers was used. These two primers (Primers 1 and 3), differed from the first set (Primers 1 and 2), in the length and position of Primer 3. From sue prediction, primers 1 and 3 were expected to generate a smaller PCR product of 146 base pairs in the case of tissues devoid of exon 8, and a larger PCR product of 155 base pairs for those containing the exon. The primers were also expected to produce more abundant PCR products, which were cntical for the ligation, subcloning and sequencing steps that followed the RT-PCR method. Therefore, RT-PCR in conjunction with primers 1 and 3 was performed on total RNA from kidney and brain. The resultiiig PCR products were electrophoresed on a 1% agarose gel, and viewed under UV illumination in order to deternine the size and abundance of the DNA frasments. The results, as indicated above, confirmed that the PCR method, using this new set of primers, did produce the expected fiagments (of 150 base pairs) which is representative of the region within the SGP-1 cDNA that was meant to be amplified (Fig. 2a). Furthemore, the two DNA bands seen in this figure are also strong, suggesting a good yield of DNA appropriate for ligation, subcloning, and sequencing. These two DNA fiagments, fiom brain and kidney, were therefore cut out of the gel and purified using the Agarose Gel Extraction Kit from Boehnnçer Mannheim.
R) C/orhg of the PCR Prorlucts The purified DNAs were ligated into the pGEMw-T Vector. The ligation products were subsequently transformed into High Efficiency Competent Cells, incubated in S.O.C. media, plated ont0 LBLX-GaVIPTG/ampicillin plates, and incubated overnight at 37°C. The results showed a good distribution of white colonies expected to contain the 150 base pair insert, compared to the distribution of blue colonies devoid of any insert (data not show). 0 Idènhicattrion of the Positive Clones To ver@ the presence of the DNA inserts, these positive white colonies were analysed using the PCR method already described. This was done in order to save tirne, by not having to grow al1 the white colonies, and not having to extract al1 the plasmid DNA before being able to sequence the sarnples. Thus, PCR was perfonned on 6 white colonies for brain and 6 for kidney, using the T7 and SP6 primers, which are specific to the promoter regions of the pGEM@-T Vector (Fig. 3). The predicted size of the PCK products containhg inserts was 300 bp, which was representative of the area amplified by the two specific pnmers. After PCR amplification, the 12 sarnples were resolved on a 1% agarose gel electrophoresis. From these results, two positive PCR products corresponding to two white colonies fiom kidney and brain, which were both the predicted length of 300 bp, were chosen for sequencing (Fig. 2b).
D) Sequencing of the KRProducts Based on these PCR resuits (Fig. 2b), the two colonies chosen for sequencing from brain and kidney were amplified in LB broth and ampicillin ovemight, and the plasrnid DNA was then separated fi-om the bacterial DNA using a plasmid DNA extraction kit fiom QIAGEN. The purified plasmid DNA was then sequenced using a sequencing gel and sequencing kit (Sequencinp Kit). The two DNA sarnples were incubated with both T7 and SP6 pnmers in separate tubes (primer annealing), combined with a special Enzyme pre-mix that contained radioactive dATP, subsequently Uicubated with sequencing mixes, and finally a Stop Solution was added to the mixture. The resulting reaction products were denatured, and then run on a sequencing gel. The gel was then dried, placed in a cassette with X-ray film for exposure, and developed (Fig. 4). The sequence of the brain sample contains the 9 base pair insertion CAG GAT CAG which represents exon 8, whereas the sequence of the kidney sarnple does not contain this insertion. Note that the sequence of the brain sample before and derthe 9 bp insertion, is identical to that of the kidney sample, and that both sequences match with the corresponding segment of the mouse SGP-1 cDNA cloned in our laboratory ( Fig. 5). 2. Analysis of the mRNA Expression of the SGP-1 Gene Total RNA fiom twelve different tissues of the mouse was extracted, run on an agarose/formaidehyde gel electrophoresis, and transferred ont0 a nylon membrane for Northem Blot analysis. The nylon membrane, containing the RNA, was then incubated with an RNA probe which was transcnbed i?om the rat cDNA encoding for the SGP-I protein, exposed to a film, and developed. The results show that there is a signal for the mRNA of SGP-1 in al1 tissues examined, indicating that they al1 contain an SGP-1 2.6 Kb mRNA transcnpt (Fig. 6).
3. Distribution of the Translated Product of the SGP-1 mRNA Eight d'ierent tissues of the mouse were dissected, fixed in Bouin's, trimrned, embedded in paraffin, sectioned. and mounted onto slides. The sections were immunostained with an anti- SGP-1 antibody (diluted 1: 100) and analysed under a light microscope. This antibody was kindly provided by Dr. Michael Gnswold from Washington State University. A) Cortex of the Brnin Cross sections of the cortex of the brain showed a strong granular reaction in the cytoplasm of al1 the cortical neurons. The nuclei of these cells were devoid of staining (Fig. 7a). B) Choroid Pims Cross sections of the choroid plexus of the fourth ventricle yielded a dark positive reaction in the cytoplasm of the epithelial cells. Their nuclei were devoid of any reaction (Fig. 7b). C) He& The anti-SGP-1 antibody produced a moderate granular reaction in the cytoplasm of the cardiocytes (Fig. 7c). D) Muscle In contrast to the heart, the anti SGP-1 antibody yielded a moderate homogeneous reaction in the cytoplasm of the muscle cells. A strong reaction was evident in the cytoplasm of some connective tissue cells. The rest of the connective tissue components were seemhgly devoid of any reaction (Fig. 7d). E) Spleen Cross sections of the spleen showed a strong reaction in the cytoplasm of macrophages surrounding the centrai vein or scattered throughout the entire organ (Fig. 7e). F3 Lung Sections of the lung parenchyma showed a strong reaction in the cytoplasm of macrophages preferentially located in the lumen of the alveoli, and in the cytoplasm of the pneumocytes type II located within the wall of the lung alveoli (Fig. 7f). G) Pancreas Cross sections of the pancreas demonstrateci a granular reaction in the perinuclear region of the a- cells. The nuclei of these cells and the zyrnogenic granules were devoid of a reaction (Fig. 7g). H) Kihq Sections of the cortex and medulla of the kidney also showed a specific staining. Most of the staining was moderate and concentrated in the cortex and partially on the proximal convoluted tubules. In general the cytoplasm of the epithelial cells of the proximal convoluted tubules were weakly stained, however, there were some isolated epithelial cells that were strongly reactive (Fig. 7h). 1-4 Te.!& Mouse testicular sections produced a strong positive reaction in the cytoplasm of Sertoli cells, regardless of the stage of the seminiferous tubule. The germ cells were devoid of any reaction. The cytoplasrn of the macrophages and Leydig cells in the interstitial spaces were also positive (Fig. 7i-j). K) Te* of ihe Rat- Positiw and Negrrtt-ve Contd Cross sections of the rat testes were used as the positive controls since this tissue has been weU documented to have a strong reaction in the Sertoli cells and a negative reaction in the geim cells with the anti-SGP- 1 antibody. Our resdts confirmed that in the semimferous tubules, the Sertoli cells were the only cells recognized by the SGP-1 antibody (Fig. 7k). A negative control was done dunng evecy irnmunostaining. This control consisted of a section of the rat testis that was immunostained with nomal rabbit serum. In al1 cases, the negative controls showed no reactive staining (data not shown), indicating that the reactions present in the other sections were solely due to the presence of SGP-1. VI. Figures adi-
Figure 1. Alternative splicing of the SGP- 1 (prosaposin) mRNA in different tissues of the mouse
Figure 2a Amplification of two DNA fragments using PCR
Figure 2b. Identification of two positive clones
Figure 3. Map of the ~GEM@-Tcloning vector
Figure 4. Sequencing of two DNA fiagrnents
Figure 5. Small portion of the cDNA sequence of mouse SGP-1
Figure 6. Regional distribution of the SGP-1 mRNA
Figure 7a-k Localization of SGP-1 in different tissues of the mouse Figure 1: Alternative spiicing of the SGP-1 mRNA in different tissues of the mouse A 12% polyacrylarnide gel electrophoresis demonstrating the alternative splicing of the SGP-1 mRNA in different tissues of the mouse determined by RT-PCR.Primer 1 and Primer 2 were used for the PCR technique. Two different sized PCR products were produced with these primers, one of 1 15 base pairs and one of 106 base pairs. Muscle (M), heart (H), and brain (B) tissues contained the larger DNA fiagment of 1 15 bp, whereas testis (T), lung (L), pancreas (P), spleen (S), and kidney O<) tissues contained the smaller 106 bp fiagment. Note a faint 106 bp band that is also present in muscle (M).
Figure 2a: Amplification of two DNA fragments using PCR A 1% agarose gel electrophoresis representing PCR products Born kidney (K) and brain (B), usiiPrimer 1 and Primer 3. The DNA ladder (La) shows that these fiagments are around 0.1 5 Kb in length and result from the PCR amplification of the region within the SGP-1 cDNA that lies between these two specific pnmers (see Fig. 5). These two DNA fragments were subcloned into the ~GEM?-TVector and subsequently sequenced.
Figure 2b: Identification of two positive u.4 ones A 1% agarose gel electrophoresis representing the amplification products of two positively cloned white bacterial colonies from brain (B) and kidney (K). PCR was performed on these colonies using the T7 and SP6 pnmers, resulting in the production of two DNA fiagments of around 0.3 Kb in length, confirming the presence of a 1 50 base pair insertion within the ~GEP-T Vector.
Figure 3: Map of the pGEM@-Tcloning vector Diagram illustrating the circle map of the pGEM@-TVector. The T7 and SP6 pnmers were used to ampli@ the region that surrounds the 150 bp DNA fi-agments fi-om Fig. 2% which were inserted into the vector. By perfonning PCR with the two specific pnmers, it was possible to confirm whether an insert was located within the vector. The predicted size of the PCR products, for colonies containhg inserts, was approximately 300 base pairs when using T7 and SP6 primers (Fig. 2b). Note the two 3l-T overhangs located at the insertion site of the 150 bp DNA fragment. They improve the efficiency ofligation of a PCR product (which contains a sinde deoxyadenosine at the 3'-end) into the plasrnid. Arnp'; ampicillin, fl ori; ongin of replication of the filamentous phage fl, lad; lac operon: promoter for the P-galactosidase gene, 3 Kb: size of the vector (Adapted fiom Promega ïèchicnl Bslletiri, 1 994).
Figure 4: Sequencing of two DNA fragments A sequencing gel was performed on the purified plasmid DNA from kidney (K) and brain (B) (Fig. 2b). The photograph shows that the sequence of the brain sample contains the 9 base pair insertion of exon 8, CAG GAT CAG (in brackets), while that of kidney does not (arrow). The sequence of the brain sample before and &er this insertion is identical to that of kidney, and both sequences match with the corresponding segment of the mouse SGP-1 cDNA that was recently cloned in our laboratory (see Fig. 5). GATCGATC
- - I) mi- Si- Figure 5: Smail Portion of the cDNA sequence of the mouse SGP-l(prosaposin) Representation of the saposin B region within the mouse SGP-1 (prosaposin) cDNA (cloned in Our laboratoty) that was amplified using RT-PCR in conjunction with two specific primers. The two arrows indicate the positions of Primer 3 (top) and Primer 1 (bottom). Nucleotide numbering are placed in the margins. The exon boundaïies are indicated with the letter (E) and the numbering identifies the exon numbers. Note that this nucleotide sequence matches the DNA sequences fi-om brain and kidney in Fig. 4. The 9 bp (CAG GAT CAG) insertion of exon 8 between bases 801 -802 is represented in bold face ( Morales et al., 1996). 667 III~~) AAC TCC AGC TTT GTC CAG GGC TCG GTG GAC CAC GTG AAG GAG GAT TGT
71 5 GAC CGC CTG GGG CCA GGC GTG TCT GAC ATA TGC AAG AAC TAC CTG GAC ..... E6 E7 ...... 763 CAG GAT CAG CAG TAT TCC GAG GTC TGT GTC CAG ATG TGG ATG CAC ATG fl CAA CCC AAG ...... E7 E9......
81 1 *III GAA ATC TGT GTG CTG GCT GGC TTC TGT AAT GAG Figure 6: Regional distribution of the SGP-1 mRNA A Northern Blot demonstrating the mRNA expression of SGP-1 in twelve different tissues of the mouse. The RNA probe used was transcribed from the rat SGP-1 cDNA. All twelve tissues show a signal for the 2.6 Kb mRNA transcript. Te; testis, Ki; kidney, He; heart, Sp; spleen, Mu; muscle, Si; smail intestine, La; large intesiine, St; stomach, Lu; lung, Br; brain, Ln; lyrnph nodes.
Figure 7a: Localization of mouse SGP-1 in the cortex of the brain Light micrograph illustrating a cross section of the cortex of the brain in the mouse, immunostained with an anti-SGP-1 antibody (1 : 100 dilution). It is evident that there is a strong reaction in the cytoplasm of al1 the cortical neurons (arrow heads). The nuclei of these cells are devoid of any reaction. X 560
Figure 7b: Localization of mouse SGP-1 in the choroid plexus of the brain Light micrograph illustrating a cross section of the choroid plexus in the brain of the mouse, immunostained with an anti SGP-1 antibody (1 :100 dilution). The cytoplasm of the epithelial cells (arrow head) lining the choroid plexus contain a dark positive reaction. X640
Figure 7c: Localization of mouse SGP-1 in the heart Light micrograph illustrating a cross section of the cardiac muscle in the heart of the mouse, immunostained with an anti-SGP-1 antibody (diluted 1: 100). A moderate granular reaction is present in the cytoplasrn of the cardiocytes. X680
Figure 7d: Localization of mouse SGP-1 in skeletal muscle Liçht micrograph illustrating a longitudinal section of the skeletal muscle in the mouse, ùnmunostained with an anti-SGP- 1 antibody (diuted 1: 100). A moderate reaction is seen in the cytoplasm of the muscle cells, whereas some comective tissue cells (arrow head) are heavily stained. The rest of the connective tissue components are devoid of any reaction. X640
Figure 7e: Localization of mouse SGP-1 in the spleen Light niicrograph of a cross section of the spleen in the mouse, imrnunostained with an anti-SGP- 1 antibody (diluted 1: 100). A strong reaction is present in the cytoplasm of the macrophages surrounding the cental vein or scattered throughout the organ. X800
Figure 7fi Localization of mouse SGP-1 in the lung Light micrograph of a cross section of the lung in the mouse, imrnunostained with an anti-SGP-1 antibody (diluted 1 : 100). The cytoplasm of the macrophages (M) are heavily stained with this antibody, and are located within the lumen of the alveoli. There is also a strong reaction present in the cytoplasm of the pneurnocytes type II cells (N) which are located within the wall of the lung alveoli. X640
Figure 7g: Localization of mouse SGP-1 in the pancreas Light micrograph illustrating a cross section of the pancreas in the mouse, immunostained with an anti-SGP-1 antibody (diluted 1 : 100). This section indicates a granular reaction in the cytoplasm of the acinar cells. The nuclei and qmogenic granules of these cells are devoid of any reaction. X520
Figure 7h: Localization of mouse SGP-1 in the kidney Light micrograph illustrating a cross section of the cortex and medulla of the kidney in the mouse, immunostained with an anti-SGP- 1 antibody (diluted 1: 100). Most of the staining is concentrated in the cortex and partially on the convoluted tubules. The cytoplasm of the epithelial cells of the proximal convoluted tubules are weakly stallied with the exception of some isolatecl cells which are strongly reactive (arrow head). X520
Figure 7i: Localization of mouse SGP-1 in the testis Light micrograph illustrating a cross section of the seminiferous tubules of the testis in the mouse, immunostained with an anti-SGP-1 antibody (diluted 1 : 100). The cytoplasm of the Sertoli cells are heavily stained (arrow heads) throughout this section. Note that the germ cells are devoid of a reaction. X 3 12
Figure 7j: Localization of mouse SGP-1 in the testis Light micrograph illustrating a cross section of t he seminiferous tubules of the testis in the mouse, immunostained with an anti-SGP-1 antibody (diluted 1: 100). Note that in this section the Sertoli cells are strongly reactive with the antibody (arrow heads), and the germ cells are devoid of a reaction. X3 12
Figure 7k: Positive control Light micrograph illustrating a cross section of the seminiferous tubules of the testis in the rat, imrnunostained with an anti-SGP- 1 antibody (diluted 1: 100). This is a positive control indicating that the anti-SGP-1 antibody used in these expenments is binding to the appropriate protein, since it has been well documented that the cytoplasrn of the Sertoli cells in the rat react positively with this particular antibody. X3 12
VIL Discussion
Regdative alternative splicing allows cells to generate different versions of a protein fiom a single gene. This mechanism results in distinct isoforrns of a protein which are expressed in different tissues, in response to the needs of the cells within that tissue (Bickrnore et al., 1992, Watson et al., 1992). The altemative splicing of the human prosaposin mRNA has been observeci by few investigators, and has recently been reported to occur in certain tissues and cells (Lamontagne and Potier 1994). The objective of the present investigation was to determine if a similar process occurs in the mouse SGP-1(prosaposin) gene, and to establish if the alternative splicing affects the production and translation of the mature mRNA. Thus, RT-PCR in conjunction with specific pnmers, Northern blot analysis, and light microscope immunocytochemistry were performed on several tissues of the mouse in order to examine these objectives.
1. Identification and Analysis of the Tissue Specific Alternative Splicing of the SGP-1 mRNA in the Mouse A) Am@fmïtion of Two DNA Fragments Using the Polymeme Chain Reacîion Alternative splicing of prosaposiiSGP-1 RNA cm lead to the production of three mRNA transcnpts, with either total inclusion of exon 8, total exclusion of exon 8 or the partial inclusion of this exon (Holtschmidt et al., 1990, Henseler et al., 1996). Reverse Transcription-Polymerase Chain Reaction (RT-PCR)has been used to identify the existence of altematively spliced human prosaposin mRNA transcnpts in three human tissues and two human ce11 lines (Lamontagne and Potier, 1994). These investigators found that the prosaposin mRNA in cultureci lymphoblasts and liver tissue did not contain the exon 8 insertion, whereas skin fibroblasts, pituitary glands, and brain contained more than 60% of mRNA with the 9 bp insertion. Furthemore, they reported that brain produced a second prosaposin mRNA transcript, which was devoid of exon 8. We employed RT-PCR to evaluate for the first time in several mouse tissues, if SGP-1 mRNA transcripts are alternatively spliced, as weU as ifthis process was tissue specific or not. Our results demonstrated that exon 8 is present in the mRNA transcript of SGP-1 in three tissues (muscle, heart, brain), and that it is absent from the transcript in the five remaining tissues (testis, lung, pancreas, spleen, and kidney). Furthermore, muscle also appeared to contain a small amount of a second mRNA transcnpt, which is devoid of the 9 bp insertion (Fig. 1). Lamontagne and Potier (1994) showed that the prosaposin mRNA transcript devoid of exon 8 was predominantly present in the white matter of the brain, whereas the mRNA with the 9 bp insertion was found in the gray matter. Our result differed hmthis finding since we found only a siieSGP-1 transcript, containing exon 8, in the brain of the mouse. Although we did not separate the gray matter from the white matter, it is possible that our sample of brain cortex contained a seemingly disproportionate amount of white and gray matter. Thus, perhaps the brain sample consisted mostly of gray matter, which would accuunt for the identification of only the exon 8 containing transcript in the brain of the mouse. Another explanation is that the other transcript is present in low concentration in mouse brain tissue, and that our assay was incapable of detecting its presence. However, RTPCR is an extremely sensitive technique, capable of ampliQing even minute amounts of RNA, and thus this contingency is unlikely. Finally, in the absence of any of these events, the possibility that the mouse and human differ in their expression of dtematively spliced prosaposidSGP-1 mRNA transcripts in brain, must be considered.
B) Cloning of the trvo DNA Fragments In order to dernonstrate that the smaller PCR product did not contain the 9 bp insertion and the larger PCR product contained the 9 bp insertion, we purified RT-PCR DNA fragments from brain and kidney, and they were ligated into pGEW-T cloning vectors. These plasmids were then used to transfomi hi@ efficiency competent cells and were subsequently plated on a special medium. AU colonies that crppear white on the plates usually contain the insert, due to the disruption of the P-galactosidase gene (Fig. 3). As this technique does yield a considerable amount of false positive results, PCR was performed on 6 colonies from brain and 6 frorn kidney to ver@ if the ~GEM"-TVector contained the insert. A sense T7 and an antisense SP6 primer were used to ampliS>the cloning site (Fig.3). We predicted that a PCR product of 300 bp should result if the amplifiecl segment contains the PCR insert, whereas only a 160 bp fiagrnent should result if the insert was not present (a fdse positive result). Two PCR products representing one white colony fi-om brain and one fiom kidney, were chosen for sequencing based on the fact that they both yielded PCR bands of 300 bp (Fig. 2b).
CJ Sequencing of the T~voDNA Fragments The sequencing of the amplified PCR products from kidney and brain produced a result that matched the sequence expected to be contained within the saposin B domain of the mouse SGP- 1 cDNA (Fig. 5). Furthemore, both sequences represented the portion of the SGP-1 cDNA surrounding the region of exon 8. The results fiom cDNA sequencing showed sue differences between the PCR products of kidney and brain (Fig. 4). The difEerence lied in the 9 bp sequence CAG GAT CAG representing the SGP-1 exon 8 that is present in brain but not in kidney. Based on these findings, we concluded that the SGP-1 mRNA of the mouse is alternatively spliced, and that this mechankm appears to be tissue specific. The results fiom the RT-PCR study described above suggest that in addition to brain, both muscle and heart transcribe rnRNAs for SGP-1 that contain the exon 8. Furthemore, the RT-PCRsuggests that the mRN A transcri pts from lung, testis, spleen, and pancreas are devoid of the exonic sequence, akin to the situation seen in the kidney. However, the PCR products of these tissues need to be sequenccd in order to confirm these conclusions. Larnontagne and Potier (1 994) performed sirnilar experiments with human tissue and ce11 culture and found that the alternative splicing of the prosaposin mRNA is also tissue specific. In order to elucidate the potential fùnclion of the alternative splicing these investigators studied the sphingolipid binding specificity of a partial synthetic peptide based on the sequence of saposin B with or without the 3 amino acid insertion encoded by exon 8. The peptide with the Gln-Asp-Gln insertion lost its capacity to bind the GM, ganglioside while its affinty for sulfatide and sphingomyelin increased significantly. These investigators proposed that these three amino acids might alter the binding specificity of saposin B towards the specific glycosphingolipids present in different tissues (Lamontagne and Potier, 1994). Thus, it is reasonable to postulate that the Gln- AspGin insertion rnight be fùnctioning in a sirnilar capacity in the SGP- 1 protein of muscle, heart, and brain of the mouse. Brain was reported to contain a greater quantity and variety of sphlngolipids. The predorninant sphingolipid in gray matter is the G,,-ganglioside whereas the white matter is rich in sulfatide (Lamontagne and Potier, 1994). The diverse sphingolipid content of brain indicates that prosaposin rnay need a more versatile binding aillnity in this tissue. This might explain why two forrns of human prosaposin mRNA, with and without exon 8, were found in human brain following RT-PCR (Lamontagne and Potier, 1994). Our results indicate the presence of two diierent rnRNA fomof SGP- 1 in muscle, suggesting that this tissue may also require saposin B with a broader spectnim of sphingolipid binding affinities. In conclusion, our results suggest that alternative splicing of mouse SGP-I rnRNA is sirnilar to the alternative splicing described for human prosaposin. It is possible that the 9 bp insertion of exon 8 may be implicated in the lysosomal or SecTetory routing of SGP-1 (prosaposin). However, our results show that in some tissues such as brain and testis, only one transcript may be expressing both the lysosomal and secretory form of this protein. Furthemore, in vivo, metabolic labelling in the testis and efferent ducts also demonstrated that SGP-1 is post-translationally modified to a 65 kDa form that is targeted to the lysosomes, and that part of the sarne protein is fùrther glycosylated to a 70 kDa form that is targeted to the extrace11ul space. Thus these results show that both proteins originate from the same transcript, suggesting that the alternative splicing of exon 8 does not affect the different routings of SGP-1. Clearly, further research in this area is necessary to elucidate the exact fûnction of the 9 bp exonic sequence coding for the 3-amino acids, Gln-Asp-Gln.
2. Regional Distribution of the SGP-1 mRNA Based on the findings that the SGP-1 mRNA is altematively spliced in different tissues of the mouse, it was important to detemine whether or not the splicing interfered with the expression of the mature mRNA transcript. In certain species, such as the Drosophila, alternative splicing of a gene has been show to affect the transcription or stability of a mature mRNA tranmipt (Baker, 1989). The sex detennination of this species depends on the alternative splicing of two important genes, resulting in the production of either Çictional RNA molecules, leading to fende development, or the splicing results in the production of non-funchonal RNA molecules in male development (Baker, 1989). In order to ensure that this was not the case of the SGP-1 RNA Northern Blot analysis was performed on 12 different tissues of the mouse using an RNA probe transcribed fiorn a rat SGP- 1 cDNA cloned in our laboratory (Morales et al., 1996). The results showed that a 2.6 Kb rnRNA transcript is present in dl 12 tissues (Fig. 6). This result also demonstrated that SGP- 1 mRNA was transcribed and stable, regardless of alternative splicing.
3. Regional and Cellular Localization of the Translated Product of the SGP-1 mRNA An imrnunocytochernical study, usiig an anti-SGP- 1 antibody, was performed in the same eight tissues exhibiting the two different mechanisms of alternative splicing of the SGP-1 gene. The objective of the study was to determine whether the altematively spliced mRNA transcripts are translated into a mature protein. The results confirmed that every tissue contains the translated protein of the SGP- I mRNq despite the form of the mRNA transcript. The reaction product was located in the cytoplasm of the cells. These findings correlate well with the notion that SGP-1 is synthesized, processed, and secreted to the extracellular space in various cellular compartments (Igdoura et al., 1996). The extracellular or secreted form of SGP-1 has been irnplicated in hctioning as a binding and transfer protein for glycosphingolipids (GSLs) Nraiwa et al., 1992). In the seminiferous tubule, SGP-1 has been reported to bind to the plasma membrane of maturing spennatids (Morales et al., 1996). It has also been reported that the secretory form of human prosaposin, the counterpart of mouse SGP-1, might be involved in the transfer of GSLs to the plasma membrane (Hiraiwa et al., 1991). Thus, the fact that the anti- SGP- 1 antibody is localized in the cytoplasm of the cells of each tissue examineci in the mouse, supports in part this notion, since the secretory routing of SGP-1, as well as its binding and transfer abilities, may also ocnir in the cytoplasm of the cell. In addition, SGP-1 is targeted to the lysosomes for its proteolytic processing into four mature saposin proteins (A-D) (Morimoto et al., 1989). Lysosomes are ubiquitous organelles within the cytoplasm of every cell (Konifeld and Mellman, 1989), which provides another explmation for the localization of SGP-1 in the cytoplasrn of cells within the different tissues of the mouse. The immunocytochernical distribution of prosaposin in the brain has been exarnined by a group of investigators, who suggrsted a possible neurotrophic function for this protein (O'Brien et al., 1994). These investigators reported that this protein is present in human cortical neurons in high concentrations. In addition, Kondoh et al. (1993) and Morales et al. (1996), also demonstrated that prosaposin is strongly immunoreactive in the neurons of the rat brain, when usbigan anti-prosapos'm antihdy. Our irnmunostaining resuhs are consistent with these findings, and indicaie that the cytoplasm of the cortical neurons of the mouse brain are heavily reactive with the ad- SGP-1 antibody, suggesting that they contain a large amount of this protein (Fig. 7a). Thus, it is possible that SGP-1 also finctions as a neurotrophic factor in the brin of the mouse, apart fiom its known fiindon as a lysosornal activator. It wodd be interesthg to study the concentrations and locations of the different isoforms of the SGP-I protein in diffkrent tissues, and to compare these results to the relative quantities and locations of the alternatively spliced rnRNA transcripts of SGP-1. However, there is still no available antibody that is able to discriminate between the prosaposin and saposin B proteins with or without the Gln-Asp-Gln insertion, encoded by exon 8. Further research on the different isoforms ofthis protein is still pending in order to understand the real function of the 3 amino acid insertion. In conclusion, our study is the first one to examine the expression of mouse SGP-1 in various tissues. It provides evidence demonstrating the presence of at least two foms of alternatively spliced transcripts, one with and one without a 9 bp insertion, encoded by exon 8 of the rnouse SGP-1 gene. Our study also demonstrated that both foms result in stable RNA molecules that translate SGP- 1. VIII. Conclusions
SGP-1 (prosaposin) exists in two forms. A 65 kDa protein which is the lysosomal precursor for four saposins, A, B, C and D which fùnction as cofactors in the degradation of glycosphingolipids (GSLs) within the lysosomes, and a 70 kDa extracellular protein present in several body fluids, and implicated as a glycolipid binding and transfer protein. The alternative splicing of the prosaposin mRNA leads to the inclusion or exclusion of a 9 bp or 6 bp insertion of exon 8, coding for Gln-Asp-Gln or Asp-Gln, respectively. This phenornenon was reported to occur in certain human tissues and ce11 lines, and was thought to be tissue specific (Lamontagne and Potier, 1994). The recent cloning of the murine SGP-1 (prosaposin) gene in our laboratory, dernonstrated that the 9 bp exon 8 is dso present. Thus, we hypothesized that alternative splicing of this gene should occur in mouse tissue. In order to test this hypothesis, we exarnined the alternative splicing of the SGP-1 gene in eight different tissues of t he mouse, using RT-PCR.The results showed thai musde, heart, and brain contained the 9 bp insertion of exon 8, whereas lung, testis, pancreas, spleen, and kidney were devoid of the insertion. We therefore concluded that alternative splicing of the SGP-1 mRN A occurs in the mouse, and that this mechanism appears to be tissue specific. Furthemore, in order to ensure that the alternative splicing of the SGP-1 rnRN A did not affect the transcription and translation of the mature mRNA transcript, Northem Blot analysis, and light microscope imunocytochemistry were perfonned in the different tissues of the mouse. Analysis of the mRNA expression of the SGP-1 gene showed the presence of a stable mRNA transcript in al1 the tissues examined, and immunocytochemical analysis of the tissue distribution of the anti-SGP- 1 antibody revealed that the altematively spliced SGP- 1 rnRNAs were translated into mature proteins. It was therefore concluded that the different foms of the SGP-1 transcripts translate SGP- 1 despite alternative splicing. Finally, it was postulated that the three amino acid insertion coded for by exon 8, may fùnction in altering the binding specificity of saposin B and SGP-1 towards specific glycosphingolipids that are present in muscle, heart, and brain tissues of the mouse. K. Original Contributions
1) This study demonstrated with the use of RT-PCR that SGP-1 rnRNA is altematively spliced in different tissues.
2) We showed for the fint tirne, that alternative splicing of the SGP-1 mRNA is different in eight tissues of the mouse, and that this rnechanism is tissue specific.
3) Northem Blot analysis showed that alternative splicing of the SGP-1 rnRNA in the mouse tissues does not affect the transcription and stability of the transcript.
4) We examined for the first time, the immunocytochemical distribution of SGP-1 in various tissues of the mouse.
5) We detemiined that regardles of alternative splicing, the different mRNA transcripts for SGP- 1 are still translated into a mature SGP- 1 protein. X. References
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