Cli. Biochem, Vol. 23, pp. 99-104. 1990 Printed in Canada. All rights reserved.
0009-9120/90 $3.00 + .00 Copyright © 1990 The Canadian Society of Clinical Chemists.
Alkaline Phosphatase Isozymes: Recent Progress WILLIAM H. FISHMAN Cancer Research Center, La Jolla Cancer Research Foundation, La Jolla, CA 92037 The past few years have witnessed the reports of significant new events in alkaline phosphatase (AP) isozymes. The cloning of the relevant genes and their nucleotide sequencing have all been accomplished. As a group, the genes for the intestinal, germ cell and placental isozymes have considerable sequence similarity; it is noteworthy that they occupy vicinal positions on chromosome 2, while the tissue unspecific AP gene is located on chromosome 1. The latter makes evolutionary lineage and instances of coordinate expression understandable. Another new development is the demonstration of a phosphatidyl inositol glycan tail on the C-terminus of these chromosome-2 AP genes. This is the major membrane insertion mechanism for AP, which is a cell surface membrane enzyme. This information may be helpful in understanding the phenomenon of the depletion of intestinal mucosal AP during fat absorption. Finally, a discussion has been focussed on recent studies on seminoma and AP, including immunodetection and immunoradiotherapy.
KEY WORDS: alkaline phosphatase; isoenzymes; alkaline phosphatase, genes; phosphatidyl inositol glycan anchor; seminoma; ovarian cancer.
Introduction ecent events have challenged old concepts of ,alkaline phosphatase (AP) isozymes and have R introduced new ideas. Reference here is to the phosphatidyl inositol mechanism for anchoring AP into the membrane. Important new information on the nature of the genes for all the isozymes, their chromosomal location and insights as to the control of their expression have also appeared. In the practical clinical enzymology arena, AP isozymes in seminomas merit discussion, while other relevant topics have been subjects of recent reviews (1-3). Two fundamental questions in Clinical Enzymology are (a) How does AP enter the circulation?; and
Correspondence: William H. Fishman, Ph.D., M.D.hc., La Jolla Cancer Research Foundation, 10901 North Torrey Pines Road, La Jolla, CA 92037, USA. Manuscript received December 21, 1988; revised June 20, 1989; accepted August 28, 1989. Abbreviations: AP = alkaline phosphatase; TUAP = tissue-unspecific alkaline phosphatase; IAP = intestinal alkaline phosphatase; GCAP = germ cell alkaline phosphatase; PLAP = placental alkaline phosphatase; PI = phosphatidyl inositol; PI-PLC = phosl~hatidyl inositol phospholipase C. CLINICAL BIOCHEMISTRY, VOLUME 23, APRIL 1990
(b) What factors control the level of circulating AP? We attempt to identify the new information, to integrate it into existing knowledge and to modify the prevailing concepts where it is justified.
H o w does alk•liue phosphatase e n t e r t h e circulation? The mechanism of AP isozyme entry into the circulation is thought to result from damage to AP rich cell surface microvilli which coat the lumens of intestine, kidney, liver, bone, placenta, etc. In addition to this mechanism, there are newly described underlying biochemical and physiological phenomena which offer alternative interpretations. The intracellular route of glycosylation of nascent enzyme protein generated in the endoplasmic reticulum has been traced from the Golgi apparatus to the cell surface membrane (4, 5). In the cell surface, AP displays lateral mobility and collects in clusters (6, 7). The physical characteristics of placental alkaline phosphatase (PLAP) isozyme molecules as measured in electron microscope studies have the dimensions of 7. 5 nm × 5.5 nm, with the shape of a rectangle enclosing a central space (8). The nucleotide sequence for PLAP cDNA predicted a hydrophobic peptide domain of some 20 amino acids at the -COOH terminus, which was initially regarded as a membrane insertion component (9-11). However, the present opinion is that AP is anchored by a phosphatidyl-inositol glycan (Figure 1) which is covalently-linked to the peptide chain (12-17). This is believed to occur via a posttranslational modification where the 29 amino acids at the -COOH terminus are exchanged for the phosphatidyl-inositol glycan component which functions as the membrane anchor. A measure of the existence of this membrane insertion linkage is its splitting by phosphatidyl-inositol specific phospholipase C (PIPLC) and the simultaneous release of the catalytically active AP (12, 13). The prior cleavage of the stretch of 29 amino acids at the -COOH terminus is believed to be mediated by a protease. The sequence of events is depicted in broad outline in Figure 2. Because it appears that only 65% of the membrane AP is released by PI-PLC, the remainder m a y be the 513 amino acid gene product anchored via the hydrophobic -COOH-terminus (12). Much remains to be done to clarify this membrane insertion phenomenon. 99
FISHMAN
484 -Asp-C-NH
III 0 ~H2 ~H2 0
I O=P-O I
0 I 6
0
Manel-2Manal\ 6
I!
Manel-4GlcNH2al-6myolnositol 1-O-P-O 3 I 0
±Gal~l-2Galel-6Galal / 2
I
I
CH2
t
±Galel
CH-O-C- CH2- (CH2) 10-CH2-CH3 0
II CH-O-C-CH2- (CH2) Io-CH2-CH3 Figure 1--Putative structure of phosphatidyl-inositol glycan.
Fat absorption and intestinal alkaline phosphatase The current picture of membrane AP provides us with a more detailed understanding of the route of intestinal AP entry into the mucosa during fat
NH2
absorption. The enrichment of the circulation with intestinal-type AP during fat absorption is a wellestablished phenomenon. Long-chain fatty acids deplete the intestinal brush border AP and, as fat particles coated with AP, travel through the mucosa into the interstitium of the lamina propria and then
PI ~
NH2 PRO-COOH
513 aa
PRO-COOH
I PI
NH2 -I-
484 aa
Figure 2--Post-translational attachment of a phosphatidyl-inositol anchor to PLAP. The carboxy terminus of 29 amino acids is replaced by phosphatidyl-inositol yielding a new carboxy terminus at aspartic acid in position 484.
100
CLINICAL BIOCHEMISTRY,VOLUME 23, APRIL 1990
ALKALINE PHOSPHATASE ISOZYMES TABLE 1 S t r u c t u r a l F e a t u r e s of Genomic A P
TUMOR MARKERS ISOENZYME PRODUCTS OF AP GENES
TUAP Introns Exons Signal peptide amino acids C-Terminus Hydrophobic domain Predicted protein domain Predicted protein M a t u r e protein Glycosylation sites Allelic v a r i a n t s
IAP
GCAP PLAP
11 12 17
10 11 19
10 11 19
?
(25)* (25) 509 (476) 2 0
(29) (29) 513 (484) 2 6
507 ? 5 0
10 11 22 29 29 513 484 2 >20
*The n u m b e r s enclosed in p a r e n t h e s e s are viewed as putative, since ASP 484 had not y e t been identified as the C - t e r m i n u s following expected phosphatidylinositol t a i l a t t a c h m e n t in the case of IAP and GCAP.
( CHROMOSOME lp 36.1- p 34
I
I
AP TISSUE UNSPECIFIC VARIANTS
I
I
I
LIVER AP BONE AP (HEPATOMA) (OSTEOGENIC SARCOMA) CHROMOSOME 2q 34.2 - q 37
I
GERM CELL PLAP AP (NAGAO) (REGAN)
lAP (KASAHARA)
Figure 3--Chromosomal locations of tumor marker isoenzyme products of AP genes (see text). into the central lacteal or lymph vessel (18, 19). It follows that the long chain triglycerides would be incorporated into the cell membrane and would readily attract the phosphatidyl inositol tail of the membrane AP into the lipid micelle. This would explain the AP coating phenomenon of the chylomicrons during fat absorption (20). Since intestinal AP in the adult lacks terminal sialic acids on its carbohydrate chains, this determines its destination to the hepatocytes via the lymph circulation. The hepatocytes take up de-sialylated glycoproteins. In this compartment, the AP has the characteristics of tissue unspecific AP undergoing inhibition by Lhomoarginine, but not by L-phenylalanine. COMMENT
The structure of the phosphatidyl-inositol glycan anchor enables one to predict that, if there is a defect in the biosynthetic mechanism for phosphatidyl inositol-alkaline phosphatase (PI-AP), the product might not anchor in the cell surface membrane. This could cause a hyperphosphatasemia which may have no relationship to any known disease. Alternatively, drugs administered for therapeutic reasons could block the biosynthetic process of PI-AP or prevent its incorporation into the cell membrane. This could also lead to hyperphosphatasemia without a clinical manifestation.
What genetic factors control .the level of circulating alkaline p h o s p h a t a s e ? It has been surmised that the genes for the individual AP isozymes are the fundamental determinants in the genesis of the protein product and its amount; factors which regulate the expression of these genes are of paramount importance. Dramatic advances have justified this concept.
CLINICAL BIOCHEMISTRY, VOLUME 23, APRIL 1990
In the past 3 years, all genes for AP isozymes have been cloned, their nucleotides sequenced, and their genomic structures elucidated. These include the genes for PLAP (9-11, 21), germ cell AP (GCAP) (22, 23), intestinal AP (IAP) (24-26) and tissue unspecific AP (TUAP) (27, 28). The structural features of genomic AP for the four isozymes are compared in Table 1. The following points are of interest. PLAP, GCAP and IAP resemble each other more than they do TUAP. They share the same number of introns and exons. The key difference is that PLAP exhibits over 20 allelic variants, GCAP six, and IAP none. The TUAP has an extra intron and exon, a shortened C-terminus, five glycosylation sites and zero allelic variants.
C h r o m o s o m a l location of the four A P g e n e s With the availability of cDNA probes, the chromosomal locations of the four AP isoenzyme genes have been established. Their relationship to each other as tumor markers is illustrated in Figure 3. Although liver and bone AP are coded by the same gene, the heat-lability of the bone variant is a distinct phenotype which has been useful in measuring the serum bone alkaline phosphatase in the circulation. Hepatomas are associated with elevated liver AP and Kasahara isozyme. The structural basis for the heat-lability of the bone AP remains to be established. It is striking that GCAP, IAP, PLAP and IAP are neighbors in the location q 34 to q 37 on chromosome 2 (29, 30). This observation now makes understandable the reciprocal expression of intestinal and placental AP isozymes when certain HeLa cell lines are exposed to corticosteroid hormones (31). The regulation of their co-expression appears to be linked on
101
FISHMAN
ANCESTRAL GENE
I
TISSUE UNSPECIFIC PRECURSOR GENE
I
INTERMEDIATE INTESTINAL AP GENE
TISSUE UNSPECIFIC AP GENE
1
J
INTESTINAL AP GENE
GERM CELL AP GENE PLAP GENE
TISSUE UNSPECIFIC AP (LIVER. BONE. EARLY PLACENTA)
INTESTINAL AP
AP
IAP
GERM CELL AP
GC-AP
f
PLAP AP
PLAP
Figure 4--Current view of the evolution of AP isozymes.
contiguous genes. On the other hand, the gene for tissue unspecific alkaline phosphatase is between p 36 and p 34 in chromosome 1 (32) and is not known to undergo reciprocal expression with IAP, GCAP and PLAP.
Hypophosphatasemia and expression of AP genes Hypophosphatasemia is a consequence of abnormal expression of the tissue unspecific AP gene or its absence, since intestinal AP is normal and transport from the intestine to the circulation is facilitated during fat absorption (20). The existence of various forms of hypophosphatasemia, each with a different genetic basis, has been reported (27). These appear to range from a missense mutation of the tissue-unspecific genelethal form of the disease to defects in the regulation of this gene (33). From the evolutionary point of view, Millan and Ryder (personal communication, 1988) propose that the germ cell AP gene appeared before the PLAP gene which is, therefore, viewed as a consequence of germ cell gene duplication and mutation. This information requires a revision of the evolutionary history of AP from that presented 2 years ago (3) (Figure 4).
Seminoma and AP isozymes It is useful at this time to concentrate on one type of cancer in which AP isozyme markers have been receiving a good deal of attention. Testicular and ovarian cancer patients exhibit the highest incidence of serum PLAP (34, 35); this correlation was notable in seminoma (36, 37). When the specificity of the serum PLAP measurements 102
was improved by a "sandwich" enzyme immunoassay (38), the clinical potential of PLAP as a tumor marker for seminoma was clearly demonstrated (39). Although the evidence shows that positive serum PLAP is found in the majority of seminoma patients, it was Uchida et al. (40), who detected the enzyme in the seminoma tissue in virtually every case. Others confirmed these observations both in sera and in tissue (41-45). Using specific monoclonal antibodies, this observation was confirmed by quantitative immunoassays (46-48). It has been known since 1980 that normal h u m a n testis contains an AP enzyme (49) with the properties of the Nagao isozyme. Thus it was heat-stable and inhibited by L-leucine. The expression of this PLAP-like isozyme (now identified as the product of the GCAP gene) was thought to be an eutopic phenomenon, representing the expression of the testicular GCAP gene, instead of the PLAP gene. Millan et al. (50) predicted that there was a specific gene locus for the testis-specific AP; this prediction was recently proven by the cloning and sequencing of germ cell AP (22). An interesting consideration in the clinical monitoring of seminoma patients arises from the work of Hirano et al. (48). Using highly specific monoclonal antibody ELISA assays for TUAP, IAP and PLAPlike isozymes, they observed that both T U A P and GCAP levels were elevated 10- to 100-fold in seminoma tissue compared to normal testis. The lower increase in IAP ranged from two- to 10-fold. There would seem to be some connection that can easily take place between chromosomes i and 2 and the regulation of their AP genes. A promising direction for greatly increasing the specificity of measurement of GCAP was reported by Millan and Manes (22). They selected a nine-amino CLINICAL BIOCHEMISTRY, VOLUME 23, APRIL 1990
ALKALINE PHOSPHATASEISOZYMES acid sequence derived from exon 11 registering two mutations of the PL A P sequence and one m ut at i on of the IAP sequence and synthesized it. When coupled to a carrier protein, it generated polyclonal antibodies in rabbits which reacted with seminoma AP, but not with PL A P in Western blots. However, the native GCAP molecule would appear to require unfolding in order to participate in a classical antigen-antibody reaction. The clinical interest in testicular and ovarian cancer is cu r r en t l y directed to the use of polyclonal and monoclonal antibodies labeled with appropriate radioisotopes both for immunodetection and immunoradiotherapy (51-53). With the promise of identifying and synthesizing key sequences of the isozyme to yield g r eater specificity, the prospect ahead is for f u r t h e r significant advances in diagnosis and therapy of these gonadal and other cancers. References
1. Fishman WH. Oncotrophoblast gene expression: Placental alkaline phosphatase. Adv Cancer Res 1987; 48: 1-35. 2. Millan JL. Oncodevelopmental expression and structure of alkaline phosphatase genes. Anticancer research 1988; 8: 995-1004. 3. Fishman WH. Clinical and biological significance of an isozyme tumor marker-PLAP. Clin Biochem 1987; 20: 387-92. 4. Hanford WC, Fishman WH. Measurement of biosynthetic rates and intracellular transit times for a cellsurface membrane glycoprotein, alkaline phosphatase in HeLa cells. Anal Biochem 1983; 129: 176-83. 5. Tokumitsu SI, Fishman WH. Alkaline phosphatase biosynthesis in the endoplasmic reticulum and its transport through the Golgi apparatus to the plasma membrane; cytochemical evidence. J Histochem Cytochem 1983; 31: 647-55. 6. Noda M, Yoon K, Rodan GA, Koppel DE. High lateral mobility of endogenous and transfected alkaline phosphatase: a phosphatidylinositol-anchored membrane protein. J Cell Biol 1987; 105: 1671-7. 7. Jemmerson R, Klier FG, Shah N, Takeya M, Fishman WH. Clustered distribution of placental-like alkaline phosphatase on the surface of A431 human epidermoid carcinoma cells: electron microscopic observations using gold-labeled antibodies. JHistochem Cytochem 1985; 33: 1227-34. 8. Takeya M, Klier FG, Fishman WH. Molecular dimensions of the SS and FF phenotypes of human placental alkaline phosphatase: rotary shadowing and negative staining electron microscope measurements. J Mol Biol 1984; 173: 253-64. 9. Kam W, Clauser E, Kim YS, Kan YW, Rutter WJ. Cloning, sequencing and chromosomal localization of human term placental alkaline phosphatase cDNA. Proc Natl Acad Sci USA 1985; 82: 8715-9. 10. Millan JL. Molecular cloning and sequence analysis of human placental alkaline phosphatase. J Biol Chem 1986; 261: 3112-5. 11. Henthorn PS, Knoll BJ, Raducha M, et al. Products of two common alleles at the locus for human placental alkaline phosphatase differ by seven amino acids. Proc Natl Acad Sci USA 1986; 83: 5597-601. 12. Jemmerson R, Low MG. Phosphatidylinositol anchor CLINICAL BIOCHEMISTRY,VOLUME 23, APRIL 1990
of HeLa cell alkaline phosphatase. Biochemistry 1987; 26: 5703-9. 13. Howard AD, Berger J, Gerber L, Familletti P, Udenfriend S. Characterization of the phosphatidylinositolglycan membrane anchor of human placental alkaline phosphatase. Proc Natl Acad Sci USA 1987; 84: 60559.
14. Takami N, Ogata S, Oda K, Misumi Y, Ikehara Y. Biosynthesis of placental alkaline phosphatase and its post-translational modification by glycophospholipid for membrane anchoring. J Biol Chem 1988; 263: 3016-21. 15. Bailey CA, Howard A, Micanovic R, et al. Site-directed antibodies for probing the structure and biogenesis ofphosphatidylinositolglycan-linked membrane proteins: application to placental alkaline phosphatase. Anal Biochem 1988; 170: 532-41. 16. Webb PD, Toss J. Attachment of human placentaltype alkaline phosphatase via phosphatidylinositol to syncytiotrophoblastand tumor cell plasma membranes. Eur J Biochem 1988; 172: 647-52. 17. Micanovic R, Bailey CA, Brink L, et al. Aspartic acid-484 of nascent placental alkaline phosphatase condenses with a phosphatidylinositol glycan to become the carboxyl terminus of the mature enzyme. Proc Natl Acad Sci USA 1988; 85: 1398--402. 18. Rufo MB, Malagelada JR, Linscheer WG, Fishman WH. Metabolic variants of intestinal alkaline phosphatase in relation to fat absorption: in situ demonstration with the organ-specific inhibitors of L-phenylalanine and L-homoarginine. Histochemie 1973; 33: 313-22. 19. Malagelada JR, Linscheer W, Rufo M, Fishman WH. Two directional release of brush border alkaline phosphatase during fat absorption. Gastroenterology 1971; 60:693 (Abstract). 20. Warshaw JB, Littlefield JW, Inglis NR, Fishman WH, Stolbach LL. Tissue origins of serum alkaline phosphatase in hypophosphatasia. J Clin Invest 1971; 51: 2137-42. 21. Knoll BJ, Rothblum KN, Longley M. Nucleotide sequence of the human placental alkaline phosphatase gene. J Biol Chem 1988; 263: 12020-7. 22. Millan JL, Manes T. Seminoma-derived Nagao isozyme is encoded by a germ cell alkaline phosphatase gene. Proc Natl Acad Sci USA 1988; 85: 3024-8. 23. Knoll BJ, Rothblum KN, Longley M. Two gene duplication events in the evolution of the human heatstable alkaline phosphatase. Gene 1987; 60: 267-76. 24. Berger J, Garattini E, Hua J-C, Udenfriend S. Cloning and sequencing of human intestinal alkaline phosphatase cDNA. Proc Natl Acad Sci USA 1987; 84: 695-8. 25. Henthorn PS, Raducha M, Edwards YH, et al. Nucleotide and amino acid sequences of human intestinal alkaline phosphatase: Close homology to placental alkaline phosphatase. Proc Natl Acad Sci USA 1987; 84: 1234-8. 26. Henthorn PS, Raducha M, Kadesch T, Weiss MJ, Harris H. Sequence and characterization of the human intestinal alkaline phosphatase gene. J Biol Chem 1988; 263: 12011-9. 27. Weiss MJ, Cole DEC, Ray K, et al. A missense mutation in the human liver/bone/kidney alkaline phosphatase gene causing a lethal form of hypophosphatasia. Proc Natl Acad Sci USA 1988; 85: 7666-9. 28. Weiss MJ, Kunal R, Henthorn PS, Lamb B, Kadesch T, Harris H. Structure of the human liver/bone/kidney 103
FISHMAN
29.
30. 31.
32.
33.
34. 35.
36.
37.
38. 39.
40. 41.
104
alkaline phosphatase gene. J Biol Chem 1988; 263: 12002-10. Martin D, Tucker DF, Gorman P, Sheer D, Spurr NK, Trowsdale J. The human placental alkaline phosphatase gene and related sequences map to chromosomes 2 band q37. Ann Hum Genet 1987; 51: 145-52. Griffin CA, Smith M, Henthorn PS, et al. Human placental and intestinal alkaline phosphatase genes map to 2q34-q37. A m J H u m Genet 1987; 41: 1025-34. Singer RM, Fishman WH. Specific isozyme profiles of alkaline phosphatase in prednisolone-treated human cell populations. In: Markert C.L., ed. Isozymes III. Developmental biology. Pp. 753-74. Academic Press, Inc., 1975. Smith M, Weiss MJ, Griffin CA, et al. Regional assignment of the gene for human liver/bone/kidney alkaline phosphatase to chromosome lp36.1-p34. Genomics 1988; 2: 139-43. Whyte MP, Magill HL, Fallon MD, Herrod HG. Infantile hypophosphatasia: normalization of circulating bone alkaline phosphatase activity followed by skeletal remineralization. Evidence for an intact structural gene for tissue nonspecific alkaline phosphatase. J Pediatr 1986; 108: 82-8. Nathanson L, Fishman WH. New observations on the Regan isoenzyme of alkaline phosphatase in cancer patients. Cancer 1972; 37: 1388--97. Fishman WH, Inglis NR, Vaitukaitis J, Stolbach LL. Regan isoenzyme and human chorionic gonadotrophin in ovarian cancer. Natl Cancer Inst Monogr 1974; 42: 63-73. Wahren B, Holmgren PA, Stigbrand T. Placental alkaline phosphatase, alphafetoprotein and carcinoembryonic antigen in testicular tumors. Int J Cancer 1979; 24: 749-53. Fishman WH, Krishnaswamy PR, Fishman L, Millan JL, McIntire KR. Gamma-glutamyl transferase in seminoma patients serum. In: Lehman F.G., ed. Carcino-embryonic proteins. Pp. 699-708. Amsterdam: Elsevier/North Holland Biomedical Press, 1979. Millan JL, Stigbrand T. "Sandwich" enzyme immunoassay for placental alkaline phosphatase. Clin Chem 1981; 27: 2014-8. Lange PH, Millan JL, Stigbrand T, Vessella RL, Ruoslahti E, Fishman WH. Placental alkaline phosphatase as a tumor marker for seminoma. Cancer Res 1982; 42: 32A-~ 7. Uchida T, Shimodo T, Miyata H, et al. Immunoperoxidase study of alkaline phosphatase in testicular tumor. Cancer 1981; 48: 1455-62. Jacobsen GK, Norgaard-Pedersen B. Placental alkaline phosphatase in testicular germ cell tumors and in
42.
43.
44.
45.
46.
47.
48.
49.
50. 51.
52.
53.
carcinoma in situ of the testis. Acta Microbiol Imunol Scand 1984; 92: 323-9. Koide O, Iwai S, Baba K, Iri H. Identification of testicular atypical germ cells by an immunohistochemical technique for placental alkaline phosphatase. Cancer 1987; 60: 1325-30. Hustin J, Collette J, Franchimont P. Immunohistochemical demonstration of placental alkaline phosphatase in various states of testicular development and in germ cell tumors. Int J Andr 1987; 10: 29-35. Paiva J, Damjanov I, Lange PH, Harris H. Immunohistochemical localization of placental-like alkaline phosphatase in testis and germ cell tumors using monoclonal antibodies. A m J Path 1983; U l : 156-65. Jeppsson A, Wahren B, Stigbrand T, Edsmyr F, Andersson L. A clinical evaluation of serum placental alkaline phosphatase (PLAP) in seminoma patients. Br J Urol 1983; 55: 73-8. Jeppsson A, Wahren B, Bretuner-Andersson E, Sillfversward C, Stigbrand T, Millan JL. Eutopic expression of placental-like alkaline phosphatase in testicular tumors. Int J Cancer 1984; 34: 757-61. Wahren B, Hinkula J, Stigbrand T, et al. Phenotypes of placental-type alkaline phosphatases in seminoma sera as defined by monoclonal antibodies. Int J Cancer 1986; 37: 595-600. Hirano K, Domar UM, Yamamoto H, Brehmer-Andersson EE, Wahren BE, Stigbrand T. Levels of alkaline phosphatase isozymes in seminoma tissue. Cancer Res 1987; 47: 2543-6. Chang CH, Angellis D, Fishman WH. Presence of the rare D-variant, heat-stable, placental-type alkaline phosphatase in normal human testis. Cancer Res 1980; 40: 1506-10. Millan JL, Ericsson A, Stigbrand T. A possible new locus of alkaline phosphatase expressed in human testis. Hum Genet 1982; 62: 293-5. Critchley M, Brownless S, Patten M, et al. Radionuclide imaging of epithelial ovarian tumors with iodine-123-1abelled monoclonal antibody (H317) specific for placental-type alkaline phosphatase. Clin Radiol 1986; 37: 107-12. Epenetos AA, Carr D, Johnson PM, Bodmer WF, Lavender JP. Antibody-guided radiolocalization of tumors in patients with testicular or ovarian cancer using two radioiodinated monoclonal antibodies to placental alkaline phosphatase. Br J Radiol 1986; 59: 117-25. Pawlikowska TRB, Epenetos AA. Radiolabelled monoclonal antibodies to placental alkaline phosphatase in the diagnosis and treatment of neoplasms of the testis, ovary and cervix. Adv Clin Enzymol 1986; 3: 53-59.
CLINICAL BIOCHEMISTRY,VOLUME 23, APRIL 1990
0009-9120/90 $3.00 + .00 Copyright © 1990 The Canadian Society of Clinical Chemists.
Alkaline Phosphatase Isozymes: Recent Progress WILLIAM H. FISHMAN Cancer Research Center, La Jolla Cancer Research Foundation, La Jolla, CA 92037 The past few years have witnessed the reports of significant new events in alkaline phosphatase (AP) isozymes. The cloning of the relevant genes and their nucleotide sequencing have all been accomplished. As a group, the genes for the intestinal, germ cell and placental isozymes have considerable sequence similarity; it is noteworthy that they occupy vicinal positions on chromosome 2, while the tissue unspecific AP gene is located on chromosome 1. The latter makes evolutionary lineage and instances of coordinate expression understandable. Another new development is the demonstration of a phosphatidyl inositol glycan tail on the C-terminus of these chromosome-2 AP genes. This is the major membrane insertion mechanism for AP, which is a cell surface membrane enzyme. This information may be helpful in understanding the phenomenon of the depletion of intestinal mucosal AP during fat absorption. Finally, a discussion has been focussed on recent studies on seminoma and AP, including immunodetection and immunoradiotherapy.
KEY WORDS: alkaline phosphatase; isoenzymes; alkaline phosphatase, genes; phosphatidyl inositol glycan anchor; seminoma; ovarian cancer.
Introduction ecent events have challenged old concepts of ,alkaline phosphatase (AP) isozymes and have R introduced new ideas. Reference here is to the phosphatidyl inositol mechanism for anchoring AP into the membrane. Important new information on the nature of the genes for all the isozymes, their chromosomal location and insights as to the control of their expression have also appeared. In the practical clinical enzymology arena, AP isozymes in seminomas merit discussion, while other relevant topics have been subjects of recent reviews (1-3). Two fundamental questions in Clinical Enzymology are (a) How does AP enter the circulation?; and
Correspondence: William H. Fishman, Ph.D., M.D.hc., La Jolla Cancer Research Foundation, 10901 North Torrey Pines Road, La Jolla, CA 92037, USA. Manuscript received December 21, 1988; revised June 20, 1989; accepted August 28, 1989. Abbreviations: AP = alkaline phosphatase; TUAP = tissue-unspecific alkaline phosphatase; IAP = intestinal alkaline phosphatase; GCAP = germ cell alkaline phosphatase; PLAP = placental alkaline phosphatase; PI = phosphatidyl inositol; PI-PLC = phosl~hatidyl inositol phospholipase C. CLINICAL BIOCHEMISTRY, VOLUME 23, APRIL 1990
(b) What factors control the level of circulating AP? We attempt to identify the new information, to integrate it into existing knowledge and to modify the prevailing concepts where it is justified.
H o w does alk•liue phosphatase e n t e r t h e circulation? The mechanism of AP isozyme entry into the circulation is thought to result from damage to AP rich cell surface microvilli which coat the lumens of intestine, kidney, liver, bone, placenta, etc. In addition to this mechanism, there are newly described underlying biochemical and physiological phenomena which offer alternative interpretations. The intracellular route of glycosylation of nascent enzyme protein generated in the endoplasmic reticulum has been traced from the Golgi apparatus to the cell surface membrane (4, 5). In the cell surface, AP displays lateral mobility and collects in clusters (6, 7). The physical characteristics of placental alkaline phosphatase (PLAP) isozyme molecules as measured in electron microscope studies have the dimensions of 7. 5 nm × 5.5 nm, with the shape of a rectangle enclosing a central space (8). The nucleotide sequence for PLAP cDNA predicted a hydrophobic peptide domain of some 20 amino acids at the -COOH terminus, which was initially regarded as a membrane insertion component (9-11). However, the present opinion is that AP is anchored by a phosphatidyl-inositol glycan (Figure 1) which is covalently-linked to the peptide chain (12-17). This is believed to occur via a posttranslational modification where the 29 amino acids at the -COOH terminus are exchanged for the phosphatidyl-inositol glycan component which functions as the membrane anchor. A measure of the existence of this membrane insertion linkage is its splitting by phosphatidyl-inositol specific phospholipase C (PIPLC) and the simultaneous release of the catalytically active AP (12, 13). The prior cleavage of the stretch of 29 amino acids at the -COOH terminus is believed to be mediated by a protease. The sequence of events is depicted in broad outline in Figure 2. Because it appears that only 65% of the membrane AP is released by PI-PLC, the remainder m a y be the 513 amino acid gene product anchored via the hydrophobic -COOH-terminus (12). Much remains to be done to clarify this membrane insertion phenomenon. 99
FISHMAN
484 -Asp-C-NH
III 0 ~H2 ~H2 0
I O=P-O I
0 I 6
0
Manel-2Manal\ 6
I!
Manel-4GlcNH2al-6myolnositol 1-O-P-O 3 I 0
±Gal~l-2Galel-6Galal / 2
I
I
CH2
t
±Galel
CH-O-C- CH2- (CH2) 10-CH2-CH3 0
II CH-O-C-CH2- (CH2) Io-CH2-CH3 Figure 1--Putative structure of phosphatidyl-inositol glycan.
Fat absorption and intestinal alkaline phosphatase The current picture of membrane AP provides us with a more detailed understanding of the route of intestinal AP entry into the mucosa during fat
NH2
absorption. The enrichment of the circulation with intestinal-type AP during fat absorption is a wellestablished phenomenon. Long-chain fatty acids deplete the intestinal brush border AP and, as fat particles coated with AP, travel through the mucosa into the interstitium of the lamina propria and then
PI ~
NH2 PRO-COOH
513 aa
PRO-COOH
I PI
NH2 -I-
484 aa
Figure 2--Post-translational attachment of a phosphatidyl-inositol anchor to PLAP. The carboxy terminus of 29 amino acids is replaced by phosphatidyl-inositol yielding a new carboxy terminus at aspartic acid in position 484.
100
CLINICAL BIOCHEMISTRY,VOLUME 23, APRIL 1990
ALKALINE PHOSPHATASE ISOZYMES TABLE 1 S t r u c t u r a l F e a t u r e s of Genomic A P
TUMOR MARKERS ISOENZYME PRODUCTS OF AP GENES
TUAP Introns Exons Signal peptide amino acids C-Terminus Hydrophobic domain Predicted protein domain Predicted protein M a t u r e protein Glycosylation sites Allelic v a r i a n t s
IAP
GCAP PLAP
11 12 17
10 11 19
10 11 19
?
(25)* (25) 509 (476) 2 0
(29) (29) 513 (484) 2 6
507 ? 5 0
10 11 22 29 29 513 484 2 >20
*The n u m b e r s enclosed in p a r e n t h e s e s are viewed as putative, since ASP 484 had not y e t been identified as the C - t e r m i n u s following expected phosphatidylinositol t a i l a t t a c h m e n t in the case of IAP and GCAP.
( CHROMOSOME lp 36.1- p 34
I
I
AP TISSUE UNSPECIFIC VARIANTS
I
I
I
LIVER AP BONE AP (HEPATOMA) (OSTEOGENIC SARCOMA) CHROMOSOME 2q 34.2 - q 37
I
GERM CELL PLAP AP (NAGAO) (REGAN)
lAP (KASAHARA)
Figure 3--Chromosomal locations of tumor marker isoenzyme products of AP genes (see text). into the central lacteal or lymph vessel (18, 19). It follows that the long chain triglycerides would be incorporated into the cell membrane and would readily attract the phosphatidyl inositol tail of the membrane AP into the lipid micelle. This would explain the AP coating phenomenon of the chylomicrons during fat absorption (20). Since intestinal AP in the adult lacks terminal sialic acids on its carbohydrate chains, this determines its destination to the hepatocytes via the lymph circulation. The hepatocytes take up de-sialylated glycoproteins. In this compartment, the AP has the characteristics of tissue unspecific AP undergoing inhibition by Lhomoarginine, but not by L-phenylalanine. COMMENT
The structure of the phosphatidyl-inositol glycan anchor enables one to predict that, if there is a defect in the biosynthetic mechanism for phosphatidyl inositol-alkaline phosphatase (PI-AP), the product might not anchor in the cell surface membrane. This could cause a hyperphosphatasemia which may have no relationship to any known disease. Alternatively, drugs administered for therapeutic reasons could block the biosynthetic process of PI-AP or prevent its incorporation into the cell membrane. This could also lead to hyperphosphatasemia without a clinical manifestation.
What genetic factors control .the level of circulating alkaline p h o s p h a t a s e ? It has been surmised that the genes for the individual AP isozymes are the fundamental determinants in the genesis of the protein product and its amount; factors which regulate the expression of these genes are of paramount importance. Dramatic advances have justified this concept.
CLINICAL BIOCHEMISTRY, VOLUME 23, APRIL 1990
In the past 3 years, all genes for AP isozymes have been cloned, their nucleotides sequenced, and their genomic structures elucidated. These include the genes for PLAP (9-11, 21), germ cell AP (GCAP) (22, 23), intestinal AP (IAP) (24-26) and tissue unspecific AP (TUAP) (27, 28). The structural features of genomic AP for the four isozymes are compared in Table 1. The following points are of interest. PLAP, GCAP and IAP resemble each other more than they do TUAP. They share the same number of introns and exons. The key difference is that PLAP exhibits over 20 allelic variants, GCAP six, and IAP none. The TUAP has an extra intron and exon, a shortened C-terminus, five glycosylation sites and zero allelic variants.
C h r o m o s o m a l location of the four A P g e n e s With the availability of cDNA probes, the chromosomal locations of the four AP isoenzyme genes have been established. Their relationship to each other as tumor markers is illustrated in Figure 3. Although liver and bone AP are coded by the same gene, the heat-lability of the bone variant is a distinct phenotype which has been useful in measuring the serum bone alkaline phosphatase in the circulation. Hepatomas are associated with elevated liver AP and Kasahara isozyme. The structural basis for the heat-lability of the bone AP remains to be established. It is striking that GCAP, IAP, PLAP and IAP are neighbors in the location q 34 to q 37 on chromosome 2 (29, 30). This observation now makes understandable the reciprocal expression of intestinal and placental AP isozymes when certain HeLa cell lines are exposed to corticosteroid hormones (31). The regulation of their co-expression appears to be linked on
101
FISHMAN
ANCESTRAL GENE
I
TISSUE UNSPECIFIC PRECURSOR GENE
I
INTERMEDIATE INTESTINAL AP GENE
TISSUE UNSPECIFIC AP GENE
1
J
INTESTINAL AP GENE
GERM CELL AP GENE PLAP GENE
TISSUE UNSPECIFIC AP (LIVER. BONE. EARLY PLACENTA)
INTESTINAL AP
AP
IAP
GERM CELL AP
GC-AP
f
PLAP AP
PLAP
Figure 4--Current view of the evolution of AP isozymes.
contiguous genes. On the other hand, the gene for tissue unspecific alkaline phosphatase is between p 36 and p 34 in chromosome 1 (32) and is not known to undergo reciprocal expression with IAP, GCAP and PLAP.
Hypophosphatasemia and expression of AP genes Hypophosphatasemia is a consequence of abnormal expression of the tissue unspecific AP gene or its absence, since intestinal AP is normal and transport from the intestine to the circulation is facilitated during fat absorption (20). The existence of various forms of hypophosphatasemia, each with a different genetic basis, has been reported (27). These appear to range from a missense mutation of the tissue-unspecific genelethal form of the disease to defects in the regulation of this gene (33). From the evolutionary point of view, Millan and Ryder (personal communication, 1988) propose that the germ cell AP gene appeared before the PLAP gene which is, therefore, viewed as a consequence of germ cell gene duplication and mutation. This information requires a revision of the evolutionary history of AP from that presented 2 years ago (3) (Figure 4).
Seminoma and AP isozymes It is useful at this time to concentrate on one type of cancer in which AP isozyme markers have been receiving a good deal of attention. Testicular and ovarian cancer patients exhibit the highest incidence of serum PLAP (34, 35); this correlation was notable in seminoma (36, 37). When the specificity of the serum PLAP measurements 102
was improved by a "sandwich" enzyme immunoassay (38), the clinical potential of PLAP as a tumor marker for seminoma was clearly demonstrated (39). Although the evidence shows that positive serum PLAP is found in the majority of seminoma patients, it was Uchida et al. (40), who detected the enzyme in the seminoma tissue in virtually every case. Others confirmed these observations both in sera and in tissue (41-45). Using specific monoclonal antibodies, this observation was confirmed by quantitative immunoassays (46-48). It has been known since 1980 that normal h u m a n testis contains an AP enzyme (49) with the properties of the Nagao isozyme. Thus it was heat-stable and inhibited by L-leucine. The expression of this PLAP-like isozyme (now identified as the product of the GCAP gene) was thought to be an eutopic phenomenon, representing the expression of the testicular GCAP gene, instead of the PLAP gene. Millan et al. (50) predicted that there was a specific gene locus for the testis-specific AP; this prediction was recently proven by the cloning and sequencing of germ cell AP (22). An interesting consideration in the clinical monitoring of seminoma patients arises from the work of Hirano et al. (48). Using highly specific monoclonal antibody ELISA assays for TUAP, IAP and PLAPlike isozymes, they observed that both T U A P and GCAP levels were elevated 10- to 100-fold in seminoma tissue compared to normal testis. The lower increase in IAP ranged from two- to 10-fold. There would seem to be some connection that can easily take place between chromosomes i and 2 and the regulation of their AP genes. A promising direction for greatly increasing the specificity of measurement of GCAP was reported by Millan and Manes (22). They selected a nine-amino CLINICAL BIOCHEMISTRY, VOLUME 23, APRIL 1990
ALKALINE PHOSPHATASEISOZYMES acid sequence derived from exon 11 registering two mutations of the PL A P sequence and one m ut at i on of the IAP sequence and synthesized it. When coupled to a carrier protein, it generated polyclonal antibodies in rabbits which reacted with seminoma AP, but not with PL A P in Western blots. However, the native GCAP molecule would appear to require unfolding in order to participate in a classical antigen-antibody reaction. The clinical interest in testicular and ovarian cancer is cu r r en t l y directed to the use of polyclonal and monoclonal antibodies labeled with appropriate radioisotopes both for immunodetection and immunoradiotherapy (51-53). With the promise of identifying and synthesizing key sequences of the isozyme to yield g r eater specificity, the prospect ahead is for f u r t h e r significant advances in diagnosis and therapy of these gonadal and other cancers. References
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