Gerald J. Mizejewski

Alpha-fetoprotein Structure and Function: Relevance to Isoforms, Epitopes, and Conformational Variants

Mammalian alpha-fetoprotein (AFP) is classified as a member of the albuminoid gene superfamily consisting of albumin, AFP, vitamin D (Gc) protein, and alpha-albumin. Molecular variants of AFP have long been reported in the biomedical literature. Early studies identified isoelectric pH isoforms and lectin-binding variants of AFP, which differed in their physicochemical properties, but not in amino acid composition. Genetic variants of AFP, differing in mRNA kilobase length, were later extensively described in rodent models during fetal/perinatal stages, carcinogenesis, and organ regeneration. With the advent of monoclonal antibodies in the early 1980s, multiple antigenic epitopes on native AFP were detected and categorized, culminating in the identification of six to seven major epitopes. During this period, various AFP-binding proteins and receptors were reported to inhibit certain AFP immunoreactions. Concomittantly, human and rodent AFP were cloned and the amino acid sequences of the translated proteins were divulged. Once the amino acid composition of the AFP molecule was known, enzymatic fragments could be identified and synthetic peptide segments synthesized. Following discovery of the molten globule form in 1981, the existence of transitory, intermediate forms of AFP were acknowledged and their physiological significance was realized. In the present review, the various isoforms and variants of AFP are discussed in light of their potential biological relevance.

Biological Roles of Alpha-Fetoprotein During Pregnancy and Perinatal Development

The use of alpha-fetoprotein (AFP) as a serum marker in cancer actually predates its employment in the detection of congenital defects; however, the latter use of AFP as a fetal defect marker has propelled its clinical utilization. Although the serum-marker capacity of AFP has long been exploited, less is known of the biological activities of this oncofetal protein during fetal and perinatal development. In the present review, the biological activities of AFP are discussed in light of this glycoproteins presence in various biological fluid compartments: embryonic and fetal tissues, serum, urine, and reproductive fluids. After a review of the histochemical detection of AFP in various cells and tissues during development, AFP concentrations within various biological fluids were discussed in the context of gestational age and anatomic location. Discussion follows concerning the relationships and roles of AFP in developmental events such as erthyropoiesis, histogenesis/organogenesis, and ligand binding and in developmental disorders such as hypothyroidism, folate deficiencies, and acquired immunodeficiency disorder (AIDS). Based on its association with so many types of birth defects, malformations, and congenital anomalies, AFP can be viewed as a molecular “troubleshooter” until signal transduction pathways are established during pregnancy and prenatal development. The review concludes with a discussion of the place of AFP in the rapidly expanding field of proteomics.

Human alpha – Fetoprotein peptides bind estrogen receptor and estradiol, and suppress breast cancer

Alpha-fetoprotein (AFP) is a transporter of various serum ligands and regulator of cellular growth during pregnancy. Estrogens modify AFP to exhibit growth suppressive properties. We recently synthesized a peptide (P149) from human AFP that suppresses the growth of mouse uterus and MCF-7 breast cancer cells. Here it is shown that molar excess treatment of native AFP with estradiol-17β (E2) exposes the P149 site on AFP. The anti-estrogenic and anti-tumor activities of AFP-peptides were tested in vivo in the immature mouse uterine assay and mammary tumor (6WI-101)-induced ascites assay, and in vitro in a cytostatic assay using five different human breast tumor cell lines. AFP-peptide P149, and fragments of P149, P149A and P149C but not P149B, suppressed the growth in both in vivo assays. P149 also suppressed the in vitro growth of MCF-7, MDA-MB-231, MDA-MB435 breast cancer cells by more than 75%. P149 and P149A bound the estrogen receptor-α (ER) with low affinities compared to E2 and tamoxifen, while P149B bound 3H-E2 with 105 fold less affinity compared to ER. The recent epidemiologic observation that high AFP levels in young pregnant women reduce their subsequent risk of postmenopausal breast cancer may be related to the growth suppressive property of AFP with the exposed P149 epitope.

ALPHA -FETOPROTEIN AS A BIOLOGIC RESPONSE MODIFIER : RELEVANCE TO DOMAIN AND SUBDOMAIN STRUCTURE

Abstract In the present review, the structure of β-fetoprotein (AFP) is discussed in consideration of AFP membership and position in the albuminoid supergene family in relation to other gene family members. Ontogenetic AFP gene expression is then discussed in view of AFP mRNA presence in various tissues at different times during development. The multiple molecular forms of AFP is also presented in relation to published reports of AFP binding proteins and cell surface receptors. The review proceeds on to present AFP as a potential model of a modular/cassette protein based on sequence comparison with cleaved fragments of prohormones and biological response modifiers. Such cleaved fragments could potentially serve as peptide messengers for vascular, neuroendocrine, and digestive biological activities. Following a discussion on fibrin binding and serine proteases, AFP-cytoskeletal, extracellular matrix, and cellular adhesion interactions are considered. AFP as a carrier/transport protein based on structural relationships is further elucidated by examination of the various ligands bound to AFP and its hormone interaction. Since AFP binds heavy metals, the question is posed of whether AFP could function as an antioxidant. An analysis of transcription factors, tumor suppressors, and homeodomain proteins follows, which is interfaced with the concept of programmed cell death in light of amino acid sequence matches detected on the AFP molecule. Emphasis was naturally placed upon the homeodomain protein sequence stretches since AFP is a fetal, phase-specific protein found throughout embryogenesis, histogenesis, and organogenesis. In keeping with histogenesis, a discussion of AFP and eye lens protein development is presented. Finally, AFP sequence analysis presented in light of members of the immunoglobulin superfamily, autoimmune disorders, and various disease states culminates the review. A closing discussion then summarizes regions of presumptive matched protein identities on each of AFPs three domains.

Physiology of Alpha-Fetoprotein as a Biomarker for Perinatal Distress: Relevance to Adverse Pregnancy Outcome:

The many physiologic roles of human alpha-fetoprotein (HAFP) and its correlation with perinatal distress/pregnancy outcome are rarely addressed together in the biomedical literature, even though HAFP has long been used as a biomarker for fetal birth defects. Although the well being of the fetus can be monitored by the measurement of gestational age–dependent HAFP in biologic fluid levels (serum, amniotic fluid, urine, and vaginal fluids) throughout pregnancy, the majority of clinical reports reflect largely second trimester and (less likely) first trimester testing due to regulatory clinical restrictions. However, reports of third-trimester and pregnancy term measurement of HAFP levels performed in clinical research and/or investigational settings have gradually increased over the years and have expanded our base knowledge of AFP-associated pregnancy disorders during these stages. The different structural forms of HAFP (isoforms, epitopes, molecular variants, etc.) detected in the various biologic fluid compartments have been limited by antibody recognition of specific epitopic sites developed by the kit manufacturers based on antibody specificity, sensitivity, and precision. Concomitantly, the advances in elucidating the various biologic actions of AFP are opening new vistas toward understanding the physiologic roles of AFP during pregnancy. The present review surveys HAFP as a biomarker for fetal distress during the perinatal period in view of its structural and functional properties. An attempt is then made to relate the AFP fluid levels to adverse pregnancy complications and outcomes. Hence, the present review was divided into two major sections: (I) AFP structure and function considerations and (II) the relationship of AFP levels to the distressed fetus during the third trimester and at term.

Purification of alpha-fetoprotein from human cord serum with demonstration of its antiestrogenic activity.

Alpha-fetoprotein (AFP) was purified from pooled human cord serum to determine whether it would be similar to purified mouse AFP in its ability to be transformed into an antiestrogen by incubation with estradiol (E2). Greater purity was attained with a three-step purification procedure of chromatofocusing, Blue-Sepharose chromatography and immunoaffinity chromatography than with a two-step procedure of polyacrylamide gel electrophoresis followed by Blue-Sepharose chromatography. Nevertheless, both procedures rendered AFP in a form that was transformable by E2 to an antiestrogen, although the product of the three-step procedure afforded more consistent biological activity. Removal of albumin from AFP was crucial for transformation of AFP to an antiestrogen. Thus, human AFP is similar to mouse AFP in being transformed to an antiestrogen upon incubation with E2, even though there is only 66% structural homology between the two proteins, and human AFP lacks the high-affinity binding site for E2 present in the mouse AFP molecule.

Interrelationships among biological activity, disulfide bonds, secondary structure, and metal ion binding for a chemically synthesized 34-amino-acid peptide derived from α-fetoprotein

A 34-amino-acid peptide has been chemically synthesized based on a sequence from human alpha-fetoprotein. The purified peptide is active in anti-growth assays when freshly prepared in pH 7.4 buffer at 0.20 g/l, but this peptide slowly becomes inactive. This functional change is proven by mass spectrometry to be triggered by the formation of an intrapeptide disulfide bond between the two cysteine residues on the peptide. Interpeptide cross-linking does not occur. The active and inactive forms of the peptide have almost identical secondary structures as shown by circular dichroism (CD). Zinc ions bind to the active peptide and completely prevents formation of the inactive form. Cobalt(II) ions also bind to the peptide, and the UV-Vis absorption spectrum of the cobalt-peptide complex shows that: (1) a near-UV sulfur-to-metal-ion charge-transfer band had a molar extinction coefficient consistent with two thiolate bonds to Co(II); (2) the lowest-energy visible d-d transition maximum at 659 nm, also, demonstrated that the two cysteine residues are ligands for the metal ion; (3) the d-d molar extinction coefficient showed that the metal ion-ligand complex was in a distorted tetrahedral symmetry. The peptide has two cysteines, and it is speculated that the other two metal ion ligands might be the two histidines. The Zn(II)- and Co(II)-peptide complexes had similar peptide conformations as indicated by their ultraviolet CD spectra, which differed very slightly from that of the free peptide. Surprisingly, the cobalt ions acted in the reverse of the zinc ions in that, instead of stabilizing anti-growth form of the peptide, they catalyzed its loss. Metal ion control of peptide function is a saliently interesting concept. Calcium ions, in the conditions studied, apparently do not bind to the peptide. Trifluoroethanol and temperature (60 degrees C) affected the secondary structure of the peptide, and the peptide was found capable of assuming various conformations in solution. This conformational flexibility may possibly be related to the biological activity of the peptide.

Alpha-fetoprotein inhibits frog metamorphosis: implications for protein motif conservation.

Alpha-fetoprotein (AFP) is a tumor-associated fetal protein which has served as a marker for both oncogenic and ontogenetic growth. A growth regulatory segment on human AFP contains amino acid sequence identity and similarity with Rana and Xenopus albumin molecules. This study assessed the ability of both intact mammalian AFP and a derived peptide to influence thyroid induction of tail resorption during Rana catesbeiana metamorphosis. After AFP and other proteins/peptides were pre-incubated with triiodothyronine (T3) for 1 h, they were added to intact tadpoles in 300 ml of water. Human and/or mouse AFP, at a concentration of 70 ng/ml, completely inhibited T3-induced tail loss when measured over a 5 day period. In contrast, albumin and other proteins were without affect. A peptide (P149) with the sequence of human AFP residues # 447-480 also completely blocked the tail response at a concentration of 33 ng/ml, whereas a scrambled version of this peptide was without activity. The present peptide segment derived from mammalian AFP might represent a highly conserved serum protein motif in the vertebrate phyla since it is capable of influencing growth, differentiation and transformation phenomenon in amphibians.

Alpha-fetoprotein (AFP)-derived peptides as epitopes for hepatoma immunotherapy: a commentary

The various immunological roles of human alpha-fetoprotein (HAFP), and its correlation with hepatomas, that is, hepatocellular carcinomas (HCCs), are not often addressed together in biomedical reports considering that HAFP is an established biomarker for hepatomas. Studies reporting measurement of HAFP serum levels in hepatoma patients in basic/clinical research settings has greatly increased over the years. Recent reports have now expanded our base knowledge in the mounting of an immune response against AFP, a self antigen, during hepatoma tumorigenesis. Advances in the detection and identification of AFP-derived peptide epitopes are opening new vistas of knowledge regarding the immunological role of AFP-peptides as T cell stimulating antigens in the course of hepatoma growth and progression. The present commentary addresses HAFP-derived peptides as immunologic responders in HCC and their use in the study and generation of AFP-peptide sensitized T cells directed against hepatoma cells. Attempts were further made to relate the AFP-derived peptide epitopes to T cell activities during the course of hepatoma immunotherapies and to profile the traits and properties of the peptides themselves. Hence, the present commentary was divided into two sections; (1) the characterization, properties, and traits of AFP peptide epitopes, and (2) the use of AFP-derived peptides in the therapeutic induction of T cells primed against hepatoma cells using both in vivo and in vitro models.

Effect of α-Fetoprotein and Derived Peptides on Insulin- and Estrogen-Induced Fetotoxicity

Both insulin and estrogen are well recognized as growth-promoting substances at physiological concentrations, but they function as teratogens at high doses. Both agents can affect alterations in fetal and maternal serum human α-fetoprotein (HAFP) levels during pregnancy. In the present study, we have employed animal models of both insulin and estrogen fetotoxicity and teratogenicity in order to study the growth-regulatory properties of HAFP and its derived peptides (HAFP/PEP). We report here the effects of HAFP/PEP on fetotoxicity, congenital malformations, and growth retardation in developing chick and murine fetuses. In the insulin model, HAFP/PEP were effective in reducing both fetal mortality and anatomic anomalies, with the result that growth-retarded fetuses were produced. With HAFP/PEP treatment, fetal demise was reduced by as much as 73 and 63% in murine and chick fetuses, respectively, while fetal anomalies were diminished by 50% during chick development. Genebank searches of identity/similarity in a HAFP/PEP fragment identified matches with a number of proteins associated with glucose, pH, ionic, osmotic, and oxidative stresses, and with heat shock, in addition to stress proteins related to protein folding/unfolding processes. It was proposed that the peptide segment on HAFP may represent a topographic ‘hotspot’, sensitive to stress/shock conditions, which exhibits a propensity for conformational alteration in the tertiary structure of the fetal protein.