Jan Frayne

Localisation of phosphatidylethanolamine-binding protein in the brain and other tissues of the rat

Abstract. Phosphatidylethanolamine-binding protein (PEBP) is a highly-conserved 21- to 23-kDa basic protein that shows preferential affinity in vitro for phosphatidylethanolamine. Previous studies have focussed on PEBP in the brain and male reproductive tract where it has been proposed to play a role in membrane biogenesis. In the present more comprehensive study, rat PEBP transcripts and protein have been found to be expressed in all tissues examined, although the levels vary considerably between tissues. However, at the cellular level, PEBP expression is enigmatic, being restricted to a diverse range of highly specialised neuronal and non-neuronal cell types. The nature of this diversity, ranging from oligodendrocytes to plasma cells, whilst not precluding a role for PEBP in membrane biogenesis in some cell types, would imply that this is not the major function in others.

The potential use of sperm antigens as targets for immunocontraception; past, present and future

Immunocontraception, and in particular the targeting of antibodies to gamete-specific antigens implicated in sperm egg binding and fertilisation, offers an attractive approach to the growing global problem of overpopulation. Such an idea is not new; indeed several immunocontraception trials, using animal model systems, have been reported in recent years and a number are reviewed here. However, the results of these studies have been largely disappointing. We believe that two fundamental flaws attribute to the poor success of most of these preliminary immunocontraceptive trials. Firstly, loss of fertility has invariably been used as the assay. This presupposes that immuno-neutralisation of a single, gamete-specific antigen will be sufficient to cause a significant reduction in fertility; however, recent data suggests that such a premise may not be well-founded for a number of reasons. Secondly, and arguably the most important flaw, is the almost universal, but largely inappropriate, use of systemic immunisation as the sole route of antigen delivery. Whilst systemic immunisation regimes may lead to high serum IgG levels, these levels do not correlate with specific antibody levels in the reproductive tract or with contraceptive efficacy. Hence, an alternative antigen delivery approach is required which will induce an effective local immune response in the reproductive tract. Here we discuss the ways in which this might be achieved.

Sequence analysis of a variety of primate fertilin α genes: Evidence for non‐functional genes in the gorilla and man

The sperm surface fertilin complex was first described in the guinea pig where it was found as a heterodimer of α and β subunits, both of which were proposed to play a role in sperm‐oolemma recognition and plasma membrane fusion during fertilisation. Whilst the β subunit is apparently testis‐specific, the finding of low levels of fertilin α in nonreproductive tissues has cast some doubt on a unique role in fertilisation. Moreover, the absence of a functional fertilin α gene in the human would imply that this gene product is not absolutely essential for fertilisation, although it could play a facilitatory role. We now describe the organisation and sequence of the fertilin α genes in a range of primates, including the great apes, and find that the gorilla gene, like that of the human, is non‐functional. Mol. Reprod. Dev. 51:92–97, 1998.

Expression of phosphatidylethanolamine‐binding protein in the male reproductive tract: Immunolocalisation and expression in prepubertal and adult rat testes and epididymides

Phosphatidylethanolamine‐binding protein (PBP) has been described previously in the male reproductive tract, where it has been implicated in the biogenesis and maintenance of antigen segregation of membranes. In the present study we have used a specific antiserum to PBP to determine its expression and localisation in the adult and prepubertal rat testis and epididymis by Western blotting and immunohistochemistry.

Rat MDC family of proteins: Sequence analysis, tissue distribution, and expression in prepubertal and adult rat testis

An increasing number of sequence‐related, cysteine‐rich membrane proteins containing metalloproteinase‐like and disintegrin‐like domains (the MDC protein family) have been identified in mammalian tissues. Here, we report the cloning and sequence analysis of cDNAs encoding several rat orthologues of this protein family, some of which are found to be expressed exclusively in the male reproductive tract, others exhibiting a broader tissue distribution. We also examine their expression in prepubertal and adult rat testis, which, in conjunction with the data on tissue distribution, form a necessary prelude to further studies aimed at establishing their individual functions. Mol. Reprod. Dev. 48:159–167, 1997.

Comparison of the proteome of adult and cord erythroid cells, and changes in the proteome following reticulocyte maturation

Cord blood stem cells are an attractive starting source for the production of red blood cells in vitro for therapy because of additional expansion potential compared with adult peripheral blood progenitors and cord blood banks usually being more representative of national populations than blood donors. Consequently, it is important to establish how similar cord RBCs are to adult cells. In this study, we used multiplex tandem mass tag labeling combined with nano-LC-MS/MS to compare the proteome of adult and cord RBCs and reticulocytes. 2838 unique proteins were identified, providing the most comprehensive compendium of RBC proteins to date. Using stringent criteria, 1674 proteins were quantified, and only a small number differed in amount between adult and cord RBC. We focused on proteins critical for RBC function. Of these, only the expected differences in globin subunits, along with higher levels of carbonic anhydrase 1 and 2 and aquaporin-1 in adult RBCs would be expected to have a phenotypic effect since they are associated with the differences in gaseous exchange between adults and neonates. Since the RBC and reticulocyte samples used were autologous, we catalogue the change in proteome following reticulocyte maturation. The majority of proteins (>60% of the 1671 quantified) reduced in abundance between 2- and 100-fold following maturation. However, ∼5% were at a higher level in RBCs, localized almost exclusively to cell membranes, in keeping with the known clearance of intracellular recycling pools during reticulocyte maturation. Overall, these data suggest that, with respect to the proteome, there is no barrier to the use of cord progenitors for the in vitro generation of RBCs for transfusion to adults other than the expression of fetal, not adult, hemoglobin.

An immortalized adult human erythroid line facilitates sustainable and scalable generation of functional red cells

With increasing worldwide demand for safe blood, there is much interest in generating red blood cells in vitro as an alternative clinical product. However, available methods for in vitro generation of red cells from adult and cord blood progenitors do not yet provide a sustainable supply, and current systems using pluripotent stem cells as progenitors do not generate viable red cells. We have taken an alternative approach, immortalizing early adult erythroblasts generating a stable line, which provides a continuous supply of red cells. The immortalized cells differentiate efficiently into mature, functional reticulocytes that can be isolated by filtration. Extensive characterization has not revealed any differences between these reticulocytes and in vitro-cultured adult reticulocytes functionally or at the molecular level, and importantly no aberrant protein expression. We demonstrate a feasible approach to the manufacture of red cells for clinical use from in vitro culture.

Induction of adult levels of β-globin in human erythroid cells that intrinsically express embryonic or fetal globin by transduction with KLF1 and BCL11A-XL

A major barrier to the clinical use of erythrocytes generated in vitro from pluripotent stem cells or cord blood progenitors is failure of these erythrocytes to express adult hemoglobin. The key regulators of globin switching KLF1 and BCL11A are absent or at a lower level than in adult cells in K562 and erythroid cells differentiated in vitro from induced pluripotent stem cells and cord blood progenitors. Transfection or transduction of K562 and cord blood erythroid cells with either KLF1 or BCL11A-XL had little effect on β-globin expression. In contrast, transduction with both transcription factors stimulated β-globin expression. Similarly, increasing the level of BCL11A-XL in the induced pluripotent stem cell-derived erythroid cell line HiDEP-1, which has levels of endogenous KLF1 similar to adult cells but lacks BCL11A, resulted in levels of β-globin equivalent to that of adult erythroid cells. Interestingly, this increase in β-globin was coincident with a decrease in ε− and ζ−, but not γ-globin, implicating BCL11A in repression of embryonic globin expression. The data show that KLF1 and BCL11A-XL together are required, but sufficient to induce adult levels of β-globin in induced pluripotent stem cell and cord blood-derived erythroid cells that intrinsically express embryonic or fetal globin.

Mutations in the second zinc finger of human EKLF reduce promoter affinity but give rise to benign and disease phenotypes

Mutations in the human erythroid Krüppel-like factor (EKLF) can lead to either anemia or the benign InLu phenotype. To elucidate the relationship between these mutations and the differing phenotypes, we prepared recombinant forms of wild-type and 5 mutant EKLF proteins and quantitated their binding affinity to a range of EKLF-regulated genes. Missense mutants (R328H, R328L, and R331G) from persons with InLu phenotype did not bind DNA. Hence, as with the heterozygous loss of function nonsense (L127X, S270X, and K292X) and frameshift (P190Lfs and R319Efs) EKLF mutations, monoallelic loss of EKLF does not result in haploinsufficiency at all loci. In contrast, K332Q has a slightly reduced DNA binding affinity (∼ 2-fold) for all promoters examined but exhibits a phenotype only in a compound heterozygote with a nonfunctional allele. E325K also has a reduced, but significant, binding affinity, particularly for the β-globin gene but results in a disease phenotype even with the wild-type allele expressed, although not as a classic dominant-negative mutant. E325K protein may therefore actively interfere with EKLF-dependent processes by destabilizing transcription complexes, providing a rational explanation for the severity of the disease phenotype. Our study highlights the critical role of residues within the second EKLF zinc finger domain.

Structure of Insoluble Rat Sperm Glyceraldehyde-3-Phosphate Dehydrogenase (Gapdh) Via Heterotetramer Formation with Escherichia Coli Gapdh Reveals Target for Contraceptive Design.

Sperm glyceraldehyde-3-phosphate dehydrogenase has been shown to be a successful target for a non-hormonal contraceptive approach, but the agents tested to date have had unacceptable side effects. Obtaining the structure of the sperm-specific isoform to allow rational inhibitor design has therefore been a goal for a number of years but has proved intractable because of the insoluble nature of both native and recombinant protein. We have obtained soluble recombinant sperm glyceraldehyde-3-phosphate dehydrogenase as a heterotetramer with the Escherichia coli glyceraldehyde-3-phosphate dehydrogenase in a ratio of 1:3 and have solved the structure of the heterotetramer which we believe represents a novel strategy for structure determination of an insoluble protein. A structure was also obtained where glyceraldehyde 3-phosphate binds in the Ps pocket in the active site of the sperm enzyme subunit in the presence of NAD. Modeling and comparison of the structures of human somatic and sperm-specific glyceraldehyde-3-phosphate dehydrogenase revealed few differences at the active site and hence rebut the long presumed structural specificity of 3-chlorolactaldehyde for the sperm isoform. The contraceptive activity of α-chlorohydrin and its apparent specificity for the sperm isoform in vivo are likely to be due to differences in metabolism to 3-chlorolactaldehyde in spermatozoa and somatic cells. However, further detailed analysis of the sperm glyceraldehyde-3-phosphate dehydrogenase structure revealed sites in the enzyme that do show significant difference compared with published somatic glyceraldehyde-3-phosphate dehydrogenase structures that could be exploited by structure-based drug design to identify leads for novel male contraceptives.