Enzymes in placental microvilli: angiotensin I converting enzyme, angiotensinase A, carboxypeptidase, and neutral endopeptidase ("enkephalinase")

Microvilli from human placental syncytiotrophoblast are rich in angiotensin I converting enzyme (ACE), aminopeptidase A, a carboxypeptidase N-like enzyme, and a neutral endopeptidase (NEP). The specific activities of these enzymes were enhanced in microvillus-enriched fractions obtained by differential centrifugation: Purified microvilli were isolated in a discontinuous sucrose gradient. The placental microvilli hydrolyzed angiotensin II, vasopressin and oxytocin as shown by high pressure liquid chromatography. The inhibitors, bestatin, phosphoramidon, and o-phenanthroline, established the specificity of the enzymes. Aminopeptidase A (angiotensinase A) cleaved angiotensin II to angiotensin III and Asp1. NEP from placenta and from human kidney hydrolyzed oxytocin at the Pro7-Leu8 bond to yield oxytocin 1-7 and leucyl-glycine amide, but did not hydrolyze vasopressin. Vasopressin was cleaved by aminopeptidases in the placental membranes. On electroblotting placental NEP appeared as a double band with a molecular weight slightly higher than the 90,000 of the purified kidney enzyme. Neuraminidase treatment reduced the molecular weight of the placental enzyme to approximately 90,000, indicating that it contains a large amount of sialic acid. The microvilli of human placenta are thus rich in enzymes that may regulate passage of peptides at the maternal-fetal interface.     

Induction by cortisol of aminopeptidases production from the human placenta in tissue culture.

Human placental aminopeptidase M and A and post-proline endopeptidase are known to act as degrading enzymes of bioactive peptides such as angiotensin II, oxytocin and endogenous opioids. We tested the effects of cortisol on the activities of human placental aminopeptidase A and M and post-proline endopeptidase using short-cultured placental tissues. From 34.5 nM to 3.45 microM of cortisol significantly increased the activities of 3 enzymes. Our present data suggest a possible important role of cortisol in the growth of human placenta via induction of placental aminopeptidases.     

Plasma S1P and Sphingosine are not Different Prior to Pre-Eclampsia in Women at High Risk of Developing the Disease

Sphingolipids like sphingosine-1-phosphate (S1P) have been implicated in the pathophysiology of pre-eclampsia. We hypothesized that plasma S1P would be increased in women at high risk of developing pre-eclampsia who subsequently develop the disease. Low circulating placental growth factor (PlGF) is known to be associated with development of pre-eclampsia; so further, we hypothesized that increased S1P would be associated with concurrently low PlGF. This was a case-control study using stored maternal blood samples from 14 to 24 weeks of pregnancy, collected from 95 women at increased risk of pre-eclampsia. Pregnancy outcome was classified as uncomplicated, preterm pre-eclampsia (<37 weeks), or term pre-eclampsia. Plasma lipids were extracted and analyzed by ultraperformance liquid chromatography coupled to electrospray ionization MS/MS to determine concentrations of S1P and sphingosine. Median plasma S1P was 0.339 nmol/ml, and median sphingosine was 6.77 nmol/l. There were no differences in the plasma concentrations of S1P or sphingosine in women who subsequently developed pre-eclampsia, no effect of gestational age, fetal sex, ethnicity, or the presence of pre-existing hypertension. There was a correlation between S1P and sphingosine plasma concentration (P < 0.0001). There was no relationship between S1P or sphingosine with PlGF. Previous studies have suggested that plasma S1P may be a biomarker of pre-eclampsia. In our larger study, we failed to demonstrate there are women at high risk of developing the disease. We did not show a relationship with known biomarkers of the disease, suggesting that S1P is unlikely to be a useful predictor of the development of pre-eclampsia later in pregnancy.     

Multiple reaction monitoring assay for pre-eclampsia related calcyclin peptides in formalin fixed paraffin embedded placenta

Although the cause of pre-eclampsia during pregnancy has not been elucidated yet, it is evident that placental and maternal endothelial dysfunction is involved. We previously demonstrated that in early onset pre-eclampsia placental calcyclin (S100A6) expression is significantly higher compared to controls ( De Groot , C. J. ; Clin. Proteomics 2007 , 1 , 325 ). In the current study, the results were confirmed and relatively quantified by using multiple reaction monitoring (MRM) on two peptide fragments of calcyclin. Cells were obtained from control (n = 5) and pre-eclamptic placental (n = 5) tissue collected by laser capture microdissection from formalin-fixed paraffin-embedded (FFPE) material treated with a solution to reverse formalin fixation. Two calcyclin peptides with an extra glycine inserted in the middle of the amino acid sequence were synthesized and used as an internal reference. Data presented show that MRM on laser microdissected material from FFPE tissue material is possible. The developed MRM assay to study quantitative levels of proteins in FFPE laser microdissected cells using nonisotopic-labeled chemical analogs of mass tagged internal references showed that in pre-eclamptic patients elevated levels of calcyclin is observed in placental trophoblast cells compared to normal trophoblast cells. By immunohistochemistry, we were able to confirm this observation in a qualitative manner.     

Gestational and smoking effects on peptidase activity in the placenta

Angiotensin converting enzyme (ACE) and leucine aminopeptidase (LAP) regulate fetally and maternally generated peptides in the placenta. In this study, ACE-like activity was found to be decreased and LAP-like activity increased with increasing days of gestation in rat placental tissues forming the fetal:maternal interface. Membrane-associated ACE-like and LAP-like activities in the placenta of smokers were also found to be significantly higher than their respective activities in placenta of nonsmokers. Our collective findings suggest that gestational and environmentally-induced changes in placental peptidase activities may account for variable peptide hormone and/or therapeutic peptide metabolism in the placenta.     

The relationship between plasma oestrone sulphate concentrations in pregnant dairy cattle and calf birth weight, calf viability, placental weight and placental expulsion

A total of 54 Holstein-Friesian cows (13 primiparous and 41 multiparous) was used to study maternal plasma oestrone sulphate (E1S) during pregnancy and its relationship to birth weight and viability of calves and time required for placental expulsion after calving. Plasma samples were obtained from the tail vein of cows once every month from days 90 to 180, every 2 weeks from days 181 to 270, and every day from day 270 of gestation to parturition. The E1S concentrations were measured by radioimmunoassay, and birth weight, placental measurements, neonatal viability and the period from calving to placental expulsion were recorded. E1S concentrations were correlated positively (0.71 > or = r > or = 0.32, P < 0.05 or P < 0.01) with calf birth weight and weights of cotyledons, intercotyledonary membranes and total placenta from days 210 of gestation to 1 day prepartum. Calf birth weight was correlated positively (p < 0.01) with the weight of the cotyledons (r = 0.87), intercotyledonary membranes (r = 0.78) and total placenta (r = 0.88). In addition, E1S concentrations were positively correlated (0.63 > or = r > or = 0.28, P < 0.05 or P < 0.01) with the neonatal viability after day 195 of pregnancy, and were negatively correlated (-0.29 > or = r > or = -0.55, P < 0.05 or P < 0.01) with the intervals from parturition to placental expulsion after 225 days of pregnancy. The results suggest that variation among dams for circulating E1S levels during late pregnancy may be caused by variation of placental development and ability for oestrogen production and conjugation, and they may influence fetal growth, neonatal viability and retained placenta.     

Predictors of ratio of placental weight to fetal weight in multiethnic community.

OBJECTIVE--To determine whether placental ratio is influenced by maternal ethnic origin, obesity, hypertension, and haematological indices of iron deficiency anaemia. DESIGN--Observational study. SETTING--District general hospital in Birmingham. SUBJECTS--692 healthy nulliparous pregnant women, of whom 367 were European, 213 Asian, 99 Afro-Caribbean, and 13 of other or undocumented ethnic origin. MAIN OUTCOME MEASURES--Placental ratio and maternal body mass index, blood pressure, and haematological indices. RESULTS--Though birth weight and placental weight were lower in Asian women than in other groups, mean placental ratio was similar in Asian (19.5% (SD 3.3%)), European (20.0% (4.0%)), and Afro-Caribbean women (20.4% (5.3%)). Gestational age at birth was the main predictor of placental ratio in the univariate analysis (r = -0.34, P < 0.001) and multivariate analysis. The only other significant predictor of placental ratio in multivariate analysis was maternal body mass index, which was positively associated with placental ratio (r = 0.1, P = 0.01). Mean (SD) placental ratio was not significantly higher in women who developed gestational hypertension (20.4% (4.5%)) and pre-eclampsia (23.3% (7.3%)) than in normal women (19.8% (3.8%)). No evidence of a relation between placental ratio and first antenatal visit haemoglobin concentration or mean cell volume was detected, and placental ratio was not associated with change in mean cell volume during pregnancy or with third trimester serum ferritin concentration. CONCLUSIONS--These data do not support the proposed association between poor maternal nutrition and increased placental ratio. The association between high placental ratio and adult hypertension may be confounded by genetic and environmental factors associated with maternal obesity (and possibly maternal hypertension).     

Laeverin/aminopeptidase Q, a novel bestatin-sensitive leucine aminopeptidase belonging to the M1 family of aminopeptidases

Laeverin/aminopeptidase Q (APQ) is a cell surface protein specifically expressed on human embryo-derived extravillous trophoblasts that invades the uterus during placentation. The cDNA cloning of Laeverin/APQ revealed that the sequence encodes a protein with 990 amino acid residues, and Laeverin/APQ contains the HEXXHX(18)E gluzincin motif, which is characteristic of the M1 family of aminopeptidases, although the exopeptidase motif of the family, GAMEN, is uniquely substituted for the HAMEN sequence. In this study, we expressed a recombinant human Laeverin/APQ using a baculovirus expression system, purified to homogeneity, and characterized its enzymatic properties. It was found that Laeverin/APQ had a broad substrate specificity toward synthetic substrate, although it showed a preference for Leu-4-methylcoumaryl-7-amide. Searching natural substrates, we found that Laeverin/APQ was able to cleave the N-terminal amino acid of several peptides such as angiotensin III, kisspeptin-10, and endokinin C, which are abundantly expressed in the placenta. In contrast to the case with other M1 aminopeptidases, bestatin inhibited the aminopeptidase activity of Laeverin/APQ much more effectively than other known aminopeptidase inhibitors. These results indicate that Laeverin/APQ is a novel bestatin-sensitive leucine aminopeptidase and suggest that the enzyme plays important roles in human placentation by regulating biological activity of key peptides at the embryo-maternal interface.     

Molecular cloning of adipocyte-derived leucine aminopeptidase highly related to placental leucine aminopeptidase/oxytocinase

In the current study, we report the cloning and initial characterization of a novel human cytosolic aminopeptidase named adipocyte-derived leucine aminopeptidase (A-LAP). The sequence encodes a 941-amino acid protein with significant homology (43%) to placental leucine aminopeptidase (P-LAP)/oxytocinase. The predicted A-LAP contains the HEXXH(X)18E consensus sequence, which is characteristic of the M1 family of zinc-metallopeptidases. Although the deduced sequence contains a hydrophobic region near the N-terminus, the enzyme localized mainly in cytoplasm when expressed in COS-7 cells. Northern blot analysis revealed that A-LAP was expressed in all the tissues tested, some of which expressed at least three forms of mRNA, suggesting that the regulation of the gene expression is complex. When aminopeptidase activity of A-LAP was measured with various synthetic substrates, the enzyme revealed a preference for leucine, establishing that A-LAP is a novel leucine aminopeptidase with restricted substrate specificity. The identification of A-LAP, which reveals strong homology to P-LAP, might lead to the definition of a new subfamily of zinc-containing aminopeptidases belonging to the M1 family of metallopeptidases.     

Effects of morphine administration and its withdrawal on rat brain aminopeptidase activities

The endogenous opioid neuropeptide system seems to be involved in the neural processes which underlie drug addiction. Several studies have reported that the administration of morphine induces changes in the levels and/or activity of endogenous opioid peptides (enkephalin, dynorphin) and their precursors in specific brain regions of the adult CNS. The aim of this work was to study the effects of chronic morphine exposure and its withdrawal on certain aminopeptidases capable of degrading opioid peptides in brain areas including the amygdala, hypothalamus, hippocampus, striatum and brain cortices. In animals treated with morphine, aminopeptidase N presented higher enzyme activity levels in the striatum, the hypothalamus and the amygdala compared to control animals, although statistically significant differences were observed only in the case of the striatum. In addition, the activity of soluble puromycin-sensitive aminopeptidase (PSA) was found to be higher in the frontal cortex of these rats. In contrast, rats experiencing withdrawal symptoms presented decreased levels of aminopeptidase activity in certain brain areas. Thus, the activity of aminopeptidase N in the hippocampus and soluble puromycin-sensitive aminopeptidase in the frontal cortex were found to be lower in rats experiencing naloxone precipitated withdrawal symptoms, compared to the corresponding controls. Finally, the activity of the three studied aminopeptidases in vitro was unaltered by incubation with morphine, suggesting that the observed effects are not due to a direct action of this opioid upon the aminopeptidases. The results of the present report indicate that aminopeptidases may play an important role in the processes of tolerance and withdrawal associated with morphine administration.     

Distribution of adipocyte-derived leucine aminopeptidase (A-LAP)/ER-aminopeptidase (ERAP)-1 in human uterine endometrium.

Adipocyte-derived leucine aminopeptidase (A-LAP, endoplasmic reticulum aminopeptidase ERAP1) is specialized to produce peptides presented on the class I major histocompatibility complex (MHC) by trimming epitopes to eight or nine residues, in addition to its enzymatic activity to degrade angiotensin II. Previously we identified placental leucine aminopeptidase (P-LAP), another member of the oxytocinase subfamily of aminopeptidases, in human uterine endometrial epithelial cells. Here we analyzed the distribution of A-LAP in human cyclic endometrium. Western blotting analysis showed that A-LAP was present in the endometrial tissue throughout the menstrual cycle. Immunohistochemical (IHC) analysis of A-LAP showed a similar distribution to that of P-LAP. A-LAP was localized predominantly in the endometrial glands and the luminal surface epithelium. However, the intracellular localization change that accompanied apocrine secretion, which was observed in P-LAP, was not shown in A-LAP. Subcellular localization of A-LAP, demonstrated by immunofluorescence, was ER in the cultured glandular epithelial cells. Our results indicate that A-LAP may fit the endometrial localization as an antigen-presenting ER aminopeptidase. Further understanding of the function(s) of A-LAP in the endometrium appear likely to yield insights into the cyclic changes during the normal endometrial cycle.     

Spontaneous oxidative DNA damage in bovine retained and nonretained placental membranes

Retention of fetal membranes (RFM) is believed to be associated with conditions of oxidative stress. In this study, 8-hydroxy-2'-deoxyguanosine (8-OH-dG) was used for the determination of spontaneous oxidative DNA lesions in maternal and fetal parts of bovine retained and nonretained placentas. Placental specimens were collected directly after spontaneous delivery or during cesarean section from cows divided into 6 groups: (A) cesarean section before term without RFM, (B) with RFM, (C) cesarean section at term without RFM, (D) with RFM, (E) spontaneous delivery at term without RFM and (F) with RFM. Isolated DNA was hydrolyzed and analyzed by HPLC; native nucleosides were monitored at 254 nm and 8-OH-dG by electrochemical detection. No significant differences in 8-OH-dG levels between retained and nonretained placental tissues were found in all samples from preterm groups (mean concentrations between 13 and 42 micromol/mol deoxyguanosine (dG)). In the term cesarean section group with RFM a significant increase in 8-OH-dG concentration in DNA from maternal (8-fold) and fetal (18-fold) membranes were detected when compared to the respective nonretained tissues. Also, in the term spontaneous delivery groups maternal nonretained placental tissues showed increased levels of 8-OH-dG in comparison to the respective tissues of the retained placenta group. In placental tissues oxidative DNA lesions appear to be controlled by responsive mechanisms which, possibly following exhaustion, give rise to increased 8-OH-dG levels.     

Effects of glucose, glycerol, 3-hydroxybutyrate, insulin, and leptin on placental growth hormone secretion in placental explants

Placental growth hormone (PGH) is secreted from the syncytiotrophoblast in increasing amounts during pregnancy. The physiology and regulation of PGH is not well known; however, low glucose levels appear to stimulate PGH liberation IN VITRO and IN VIVO. PGH appears to have lipolytic effects, and inverse correlations between maternal body mass index and serum PGH levels have been reported. Therefore, substances related to maternal adipose tissue metabolism could influence PGH secretion. The effect of insulin, glycerol, 3-hydroxybutyrate (3-OHB), and leptin on PGH and human placental lactogen (hPL) secretion from cultured placental explants was studied. In glucose-free media, PGH content increased upto 237.5+/-28.4% of control media (p<0.001, ANOVA). Insulin levels were without effect on PGH secretion, as were 3-OHB, leptin, and glycerol at 0.02 mmol/l. Glycerol at 0.2 mmol/l increased PGH in all of the placental explants studied (n=8; mean increase 27.3+/-7.1%), and this difference was significantly different from the control explants (p=0.004). The liberation of hPL to culture media was different from PGH and was influenced by glucose and insulin. In conclusion, the absence of glucose profoundly increased PGH secretion in cultured placental explants. Addition of glycerol in physiologically relatively high concentrations showed a less pronounced stimulatory effect.     

The vitamin D receptor is not required for fetal mineral homeostasis or for the regulation of placental calcium transfer in mice

We utilized a vitamin D receptor (VDR) gene knockout model to study the effects of maternal and fetal absence of VDR on maternal fertility, fetal-placental calcium transfer, and fetal mineral homoeostasis. Vdr null mice were profoundly hypocalcemic, conceived infrequently, and had significantly fewer viable fetuses in utero that were also of lower body weight. Supplementation of a calcium-enriched diet increased the rate of conception in Vdr nulls but did not normalize the number or weight of viable fetuses. Among offspring of heterozygous (Vdr(+/-)) mothers (wild type, Vdr(+/-), and Vdr null fetuses), there was no alteration in serum Ca, P, or Mg, parathyroid hormone, placental (45)Ca transfer, Ca and Mg content of the fetal skeleton, and morphology and gene expression in the fetal growth plates. Vdr null fetuses did have threefold increased 1,25-dihydroxyvitamin D levels accompanied by increased 1alpha-hydroxylase mRNA in kidney but not placenta; a small increase was also noted in placental expression of parathyroid hormone-related protein (PTHrP). Among offspring of Vdr null mothers, Vdr(+/-) and Vdr null fetuses had normal ionized calcium levels and a skeletal ash weight that was appropriate to the lower body weight. Thus our findings indicate that VDR is not required by fetal mice to regulate placental calcium transfer, circulating mineral levels, and skeletal mineralization. Absence of maternal VDR has global effects on fetal growth that were partly dependent on maternal calcium intake, but absence of maternal VDR did not specifically affect fetal mineral homeostasis.     

Identification of human aminopeptidase O, a novel metalloprotease with structural similarity to aminopeptidase B and leukotriene A4 hydrolase

We have cloned and characterized a human brain cDNA encoding a new metalloprotease that has been called aminopeptidase O (AP-O). AP-O exhibits a series of structural features characteristic of aminopeptidases, including a conserved catalytic domain with a zinc-binding site (HEXXHX18E) that allows its classification in the M1 family of metallopeptidases or gluzincins. The structural complexity of AP-O is further increased by the presence of an additional C-terminal domain 170 residues long, which is predicted to have an ARM repeat fold originally identified in the Drosophila segment polarity gene product Armadillo. This ARM repeat domain is also present in aminopeptidase B, aminopeptidase B-like, and leukotriene A4 hydrolase and defines a novel subfamily of aminopeptidases that we have called ARM aminopeptidases. Northern blot analysis revealed that AP-O is mainly expressed in the pancreas, placenta, liver, testis, and heart. Human AP-O was produced in Escherichia coli, and the purified recombinant protein hydrolyzed synthetic substrates used for assaying aminopeptidase activity. This activity was abolished by general inhibitors of metalloproteases and specific inhibitors of aminopeptidases. Recombinant AP-O also cleaved angiotensin III to generate angiotensin IV, a bioactive peptide of the renin-angiotensin pathway with multiple actions on diverse tissues, including brain, testis, and heart. On the basis of these results we suggest that AP-O could play a role in the proteolytic processing of bioactive peptides in those tissues where it is expressed.     

A new approach regarding the treatment of preeclampsia and preterm labor

Both preeclampsia and preterm delivery are important complications in pregnancy and are leading causes for maternal and perinatal morbidity and mortality. The underlying molecular mechanisms of both diseases remain unknown, thus treatments (beta2-stimulants and magnesium sulfate) are essentially symptomatic. Both molecules have molecular weights less than 5-8 kDa and cross the placental barrier thus exerting their effects on the fetus. In addition, the fetus produces peptide hormones that are highly vasoactive and uterotonic and increase in response to maternal stress and with continued development. Fetal peptides are also small molecules that inevitably leak across into the maternal circulation. Aminopeptidases such as placental leucine aminopeptidase (P-LAP) and aminopeptidase A (APA) are large molecules that do not cross the placental barrier. We have shown that APA acts as an antihypertensive agent in the pregnant spontaneously hypertensive rat by degrading vasoactive peptides and as a result returns the animal to a normotensive state. We have also noted that P-LAP acts as an anti-uterotonic agent by degrading uterotonic peptides, and as a result prolongs gestation in the pregnant mouse. Thus, P-LAP and APA represent promising agents for the treatment of preeclampsia and preterm labor by degrading bioactive hormones derived from the feto-placental circulation.     

Positioning of aminopeptidase inhibitors in next generation cancer therapy

Aminopeptidases represent a class of (zinc) metalloenzymes that catalyze the cleavage of amino acids nearby the N-terminus of polypeptides, resulting in hydrolysis of peptide bonds. Aminopeptidases operate downstream of the ubiquitin–proteasome pathway and are implicated in the final step of intracellular protein degradation either by trimming proteasome-generated peptides for antigen presentation or full hydrolysis into free amino acids for recycling in renewed protein synthesis. This review focuses on the function and subcellular location of five key aminopeptidases (aminopeptidase N, leucine aminopeptidase, puromycin-sensitive aminopeptidase, leukotriene A₄ hydrolase and endoplasmic reticulum aminopeptidase 1/2) and their association with different diseases, in particular cancer and their current position as target for therapeutic intervention by aminopeptidase inhibitors. Historically, bestatin was the first prototypical aminopeptidase inhibitor that entered the clinic 35 years ago and is still used for the treatment of lung cancer. More recently, new generation aminopeptidase inhibitors became available, including the aminopeptidase inhibitor prodrug tosedostat, which is currently tested in phase II clinical trials for acute myeloid leukemia. Beyond bestatin and tosedostat, medicinal chemistry has emerged with additional series of potential aminopeptidases inhibitors which are still in an early phase of (pre)clinical investigations. The expanded knowledge of the unique mechanism of action of aminopeptidases has revived interest in aminopeptidase inhibitors for drug combination regimens in anti-cancer treatment. In this context, this review will discuss relevant features and mechanisms of action of aminopeptidases and will also elaborate on factors contributing to aminopeptidase inhibitor efficacy and/or loss of efficacy due to drug resistance-related phenomena. Together, a growing body of data point to aminopeptidase inhibitors as attractive tools for combination chemotherapy, hence their implementation may be a step forward in a new era of personalized treatment of cancer patients.     

脐动脉血流对预测子痫前期新生儿和产妇结局的价值分析

目的:探讨应用脐动脉血流用于预测子痫前期新生儿和产妇结局的临床价值。方法:选择在我院产科建档分娩的120例孕产妇作为研究对象,根据子痫前期发病情况分为子痫前期组60例与对照组60例,记录和比较两组孕产妇的一般资料、血脂、血糖水平、分娩前脐动脉血流与新生儿体重、胎盘的重量及Apgar评分,并进行相关性与危险因素分析。结果:两组孕产妇的年龄、孕次、产次、流产次数、孕周等对比差异均无统计学意义(P0.05)。子痫前期组的血清HDL-C水平低于对照组(P0.05),血清TC、TG、LDL-C、FBG水平高于对照组(P0.05)。与对照组比较,子痫前期组脐动脉S/D、RI与PI值显著升高(P0.05)。所有孕产妇都顺利完成分娩,孕产妇与新生儿都存活,子痫前期组的新生儿出生体重及Apgar评分和胎盘的重量均显著低于对照组(P0.05)。在子痫前期组中,脐动脉S/D、RI、PI值与新生儿出生体重呈现显著负相关性(P0.05)。多重线性回归分析显示子痫前期孕产妇的脐动脉S/D、RI、PI值为影响新生儿出生体重的独立危险因素(P0.05)。结论:脐动脉血流与子痫前期新生儿出生体重显著相关,脐动脉S/D、RI、PI值为影响新生儿出生体重的独立危险因素,子痫前期脐动脉血流监测可为预测新生儿和产妇结局以及预后提供参考。     

Analysis of the role of bleomycin hydrolase in antigen presentation and the generation of CD8 T cell responses

Long oligopeptides (>10 residues) are generated during the catabolism of cellular proteins in the cytosol. To be presented to T cells, such peptides must be trimmed by aminopeptidases to the proper size (typically 8-10 residues) to stably bind to MHC class I molecules. Aminopeptidases also destroy epitopes by trimming them to even shorter lengths. Bleomycin hydrolase (BH) is a cytosolic aminopeptidase that has been suggested to play a key role in generating MHC class I-presented peptides. We show that BH-deficient cells from mice are unimpaired in their ability to present epitopes from N-extended precursors or whole Ags and express normal levels of MHC class I molecules. Similarly, BH-deficient mice develop normal CD8(+) T cell responses to eight epitopes from three different viruses in vivo. Therefore, BH by itself is not essential for the generation or destruction of MHC class I peptides. In contrast, when BH(-/-) mice are crossed to mice lacking another cytosolic aminopeptidase, leucine aminopeptidase, the resulting BH(-/-)leucine aminopeptidase(-/-) progeny show a selective increase in CD8(+) T cell responses to the gp276 epitope from lymphocytic choriomeningitis virus, whereas the ability to present and respond to several other epitopes is unchanged. Therefore, BH does influence presentation of some Ags, although its role is largely redundant with other aminopeptidases.     

Characterization of a recombinant soluble form of human placental leucine aminopeptidase/oxytocinase expressed in Chinese hamster ovary cells.

Serum levels of human placental leucine aminopeptidase/oxytocinase (P-LAP) increase with gestation. cDNA cloning of P-LAP revealed that the enzyme is a type II membrane-bound protein containing the consensus HEXXH(X)18E motif found in the M1 family of zinc-metallopeptidase proteins. In this study, a recombinant soluble form of P-LAP found in maternal serum was expressed in Chinese hamster ovary cells, purified to homogeneity and then characterized. Although N-terminal sequencing revealed a four-amino-acid deletion, the purified enzyme was active and was shown to be a zinc-containing homodimeric protein with molecular mass of 280 kDa in solution. Using artificial substrates, it was shown that the enzyme has broad specificity and is inhibited by several compounds known as aminopeptidase inhibitors. Subsequently, sequential N-terminal amino-acid liberation of several peptide hormones by the enzyme was monitored and structures of the products were determined. Among the hormones having a cysteine residue at their N-terminal end and intramolecular disulfide bonds, it was found that vasopressin and oxytocin, but not calcitonin and endothelins, were cleaved by the enzyme. Because the molecular properties of oxytocinase so far reported often conflict, our results provide an initial biochemical and enzymatic characterization of moleculary defined P-LAP/oxytocinase.