Abstract
A sensitive and specific enzyme-linked immunoabsorbent assay was used in a retrospective study of serum levels of placental alkaline phosphatase (PLAP) in testicular cancer. Sixteen of 28 men with active seminoma had elevated PLAP levels, and 71% had elevated levels of either PLAP, human chorionic gonadotropin, or both. Only four of 22 men with active nonseminomatous cancer had elevated PLAP levels, and the levels were normal in all control patients, including 33 men apparently cured of testicular cancer. In six of ten serial studies, PLAP levels provided information not otherwise available that would have been useful clinically, and the levels never were elevated inappropriately. Our data suggest that PLAP is a clinically useful serum tumor marker for seminoma.
Introduction
Alkaline phosphatases [ALP; orthophosphoric monoester phosphohydrolase (alkaline optimum) EC 3.1.3.1] are plasma membrane-bound glycoproteins [1, 2]. These enzymes are widely distributed in nature, including prokaryotes and higher eukaryotes [3–6], with the exception of some higher plants [7]. Alkaline phosphatase forms a large family of dimeric enzymes, usually confined to the cell surface [8, 9] hydrolyzes various monophosphate esters at a high pH optimum with release of inorganic phosphate [10, 11].
Mammalian alkaline phosphatases (ALPs) are zinc-containing metalloenzymes encoded by a multigene family and function as dimeric molecules. Three metal ions including two Zn2+ and one Mg2+ in the active site are essential for enzymatic activity. However, these metal ions also contribute substantially to the conformation of the ALP monomer and indirectly regulate subunit–subunit interactions
Isoforms of Alkaline Phosphatase and Their Distribution
Human ALPs can be classified into at least four tissue-specific forms or isozyme mainly according to the specificity of the tissue to be expressed, termed as placental alkaline phosphatase (PLALP or Regan isozyme), Intestinal alkaline phosphatase (IALP), liver/bone/kidney alkaline phosphatase (L/B/K ALP), germ cell ALP (GCALP or NAGAO isozyme) [13]. The enzyme products of at least three ALP gene loci (placental, intestinal and L/B/K) [14–16] are distinguishable in man by a variety of structural, biochemical and immunologic methods [17–19].
Placental Alkaline Phosphatase
The human placental ALP gene was mapped to chromosome 2 [20]. A homology of 87 % is found with the IAP gene. There are, however, differences at their carboxyl terminal end [21]. Placental ALP is a heat stable enzyme present at high levels in the placenta. A trace amount of this isoenzyme can be detected in normal sera [22]. Part of the serum placental-type activity originates from neutrophils. The placental ALP gene can be re-expressed by cancer cells as the Regan isoenzyme. Placental ALP is a polymorphic enzyme, with up to 18 allelozymes resulting from point mutations, in contrast to the other ALP isoenzymes [23].
Intestinal Alkaline Phosphatase
The gene encoding for intestinal ALP (IAP) is a member of the gene family mapping to the long arm of chromosome 2 [24]. IAP is partially heat-stable isozyme present at high levels in intestinal tissue. In contrast to the other ALP isoenzymes, the carbohydrate side-chains of IAP are not terminated by sialic acid [25]. Distinct IALPs can be isolated from fetal and adult intestinal tissue, with the fetus forming a sialylated isoenzyme in contrast to the adult. The fetal and adult forms differ not only in the carbohydrate content but also in the protein moiety itself, suggesting that a separate ALP gene locus may exist in humans during fetal development. This embryonic gene can be reexpressed (in a modified form) by cancer cells and is designated as Kasahara isoenzyme [26].
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Germ Cell Alkaline Phosphatase
The gene encoding for germ-cell ALP (GCAP, placental-like ALP) was also mapped to chromosome 2 [27]. It is heat-stable isozyme present at low levels in germ cells [2] embryonal and some neoplastic tissues [28, 29]. It encodes testis/thymus ALP and can be expressed in the placenta at low levels [30]. GCAP in testis appears to be localized to the cell membrane of immature germ cells and, like the other ALP isoenzymes, is attached to the cell membrane by means of a phosphatidyl-inositol-glycan anchor. Like the placental ALP gene, it can be reexpressed by cancer cells (or NAGAO isozyme) [31].
Liver/Bone/Kidney Alkaline Phosphatase
The heat-labile isozyme represents the liver/bone/kidney or tissue nonspecific (TNSALP) form [1, 2]. It is expressed in many tissues throughout the body and is especially abundant in hepatic, skeletal, and renal tissue. Slight differences in electrophoretic mobility and thermo stability between the L/B/K ALPs from various tissues are attributed to differences in post-translational modification, although it is possible that their protein moieties are encoded by separate but related genes [19].
Liver/bone/kidney or tissue-nonspecific alkaline phosphatase (TNSALP) is encoded as a single genetic locus, mapped to the short arm of chromosome 1 [32, 33]. Komoda and Sakagishi [25] postulated a hypothesis regarding the physiological role of the sugar moieties in ALPS: they would protect the enzyme from rapid removal from the circulation through binding by the asialoglycoprotein receptors of the liver.
The evolution of the ALP gene family has presumably involved the duplication of a primordial tissue-nonspecific ALP gene to create the TNSALP gene and an intermediate IAP gene, followed by additional duplications of the latter to create intestinal, placental, and germ-cell ALP genes. Only humans and great apes have placental ALP; all other mammals have IAP.
Structure of the Gene
Alkaline phosphatase is a membrane-bound metalloenzyme that consists of a group of isoenzymes. Each isoenzyme is a glycoprotein encoded by different gene loci. It is believed that all of the human ALP genes evolved from a single ancestral gene. shows a rough outline of the deduced evolutionary tree of ALP
Placental alkaline phosphatase (PLAP) is an enzyme encoded by the ALPP gene. Alkaline phosphotases (ALPases) are membrane-associated sialoglycoprotein enzymes, which are normally present at high concentration in syncytiotrophoblasts of the placenta during the third trimester of gestation. PLAP has only been observed in Hominidae (greater apes) and is very different from the ALPases present in lower primates and mammals. The duplication event resulting in the appearance of human-type placental ALPase occurred subsequent to the divergence of the evolutionary lineage leading to the Hylobatidae (lesser apes), but prior to the divergence of the lineage leading to the Hominidae. Also, a number of changes to its promoter region resulted in the subsequent enzyme’s placental specific expression. Human PLAP has several variants of which three are polymorphic, although the importance and possible selective advantages are not yet explored.
Background Information:
Placental ALPase, produced by the fetal side of the placenta, is anchored, in a homodimeric form, to the apical and basal plasma membranes of syncytiotrophoblasts. PLAP expression increases steadily throughout pregnancy until term when it decreases post-partum, and is released in maternal serum starting the second trimester of gestation. The physiological role of placental ALPase is still unclear. As PLAP is Hominidae-specific, studies in other primates might provide insights into higher primate evolution.
The Human Difference:
Lesser apes and other primates do not have an isoenzyme that resembles human placental phosphatase. The placental enzyme of these species more closely resembles the human liver, bone, and kidney types of ALPase. Recent evidence suggests that the emergence of placental expression of PLAP is prior to the divergence of lineages leading to he Hominidae, but after the lineages leading to lesser apes, like gibbon. It is unclear, however, if the duplication occurred within this timeframe or only the change in regulatory mechanism. An old world monkey lung-specific ALPase could be the product of the same duplication event, but diverged in expression within the two lineages. A comparison of the placental ALP’s 5’ flanking sequence (up to -540) with that of intestinal ALP gene shows many deletion/substitutions which could result in tissue-specific expression changes.
Universality in Human Populations:
All humans express PLAP. Unlike most ALPases, which are highly conserved, the PLAP locus is variable, with three polymorphic and at least 15 rare variants documented. Three of these alleles, named Pl1, Pl2 and Pl3, give rise to six common electrophoretic phenotypes characterized by different enzyme activities. PI1 and PI2 display differential mRNA expression linked to a sequence variation within their promoter regions. The relevance of these variants is not well studied.
