Cryo-EM Structure of Human Hyaluronidase PH-20.

Ligand-receptor signaling with the epidermal growth factor (EGF) family of growth factors in the uterus and embryo is considered to be important for implantation. The EGF family includes EGF, transforming growth factor-α, heparin binding-EGF, amphiregulin, betacellulin, epiregulin, and heregulins, whereas the receptor family (the erbB genes) consists of erbB1 (EGF-receptor, EGF-R), erbB2,erbB3, and erbB4. Interactions of uterine EGF-R with EGF-like ligands have been examined, but limited information is available regarding the status of other receptor subtypes. Thus, we examined the expression of the erbB2 gene in the mouse uterus during the periimplantation period (days 1–8 of pregnancy) and after 17β-estradiol and/or progesterone stimulation. Northern blot hybridization detected two transcripts (∼4.0 and 5.0 kb) of erbB2 messenger RNA (mRNA) in day 1–8 uterine polyadenylated RNA samples. In situ hybridization experiments showed unique uterine cell-specific erbB2 mRNA distribution. On days 1–4, unlike the full-length erbB1 mRNA which is not expressed in the uterine epithelium, the erbB2 mRNA was detected primarily in epithelial cells; the day 1 uterus showed the highest accumulation. On day 5, the epithelium and the decidualizing stromal cells around the implanting blastocyst exhibited accumulation of this mRNA. On days 6–8, the accumulation persisted in the epithelium at both the implantation and interimplantation sites in addition to modest levels of signals in the secondary decidual zone. On days 7 and 8, accumulation of the erbB2 mRNA was also prominent in the trophoblastic giant cells. Western blotting detected a predicted protein of 185 kDa in day 4 uterine membrane preparations. Results of immunocytochemistry demonstrated colocalization of the erbB2 protein with its mRNA in the periimplantation uterus. The uterine ErbB2 underwent phosphorylation by several members of the EGF family. Treatment of adult ovariectomized mice with 17β-estradiol, but not progesterone, up-regulated the expression of the erbB2 mRNA by more than 3.5-fold, as determined by quantitative reverse transcription-PCR, and this increase was limited to the epithelium, as revealed by in situ hybridization. Collectively, the results place ErbB2 as a potential candidate receptor subtype for interaction with the EGF-related ligands in epithelial cell proliferation/differentiation during the preimplantation period and stromal cell proliferation/decidualization during the postimplantation period.

Epidermal growth factor family and renal cell carcinoma: expression and prognostic impact.

Thrombomodulin (TM) increases the catalytic efficiency of thrombin (IIa)-mediated activation of thrombin-activable fibrinolysis inhibitor (TAFI) 1250-fold. Negatively charged residues of the C-loop of TM-EGF-like domain 3 are required for TAFI activation. Molecular models suggested several positively charged residues of TAFI with which the C-loop residues could interact. Seven TAFI mutants were constructed to determine if these residues are required for efficient TAFI activation. TAFI wild-type or mutants were activated in the presence or absence of TM and the kinetic parameters of TAFI activation were determined. When the three consecutive lysine residues in the activation peptide of TAFI were substituted with alanine (K42/43/44A), the catalytic efficiencies for TAFI activation with TM decreased 8-fold. When other positively charged surface residues of TAFI (Lys-133, Lys-211, Lys-212, Arg-220, Lys-240, or Arg-275) were mutated to alanine, the catalytic efficiencies for TAFI activation with TM decreased by 1.7-2.7-fold. All decreases were highly statistically significant. In the absence of TM, catalytic efficiencies ranged from 2.8-fold lower to 1.24-fold higher than wild-type. None of these, except the 2.8-fold lower value, was statistically significant. The average half-life of the TAFIa mutants was 8.1+/-0.6 min, and that of wild type was 8.4+/-0.3 min at 37 degrees C. Our data show that these residues are important in the activation of TAFI by IIa, especially in the presence of TM. Whether the mutated residues promote a TAFI-TM or TAFI-IIa interaction remains to be determined. In addition, these residues do not influence spontaneous inactivation of TAFIa.

The ligand-mediated down-regulation of the growth factor receptors is preceded by the involvement of various other factors. In particular, a ubiquitin ligase, Cbl, plays a central role in this event. Several candidates that have potential effects on the negative control of the epidermal growth factor (EGF) receptor have now been identified by our recent studies in phospho-proteomics. Among these molecules, we focus on characterizing a novel protein, Ymer, which is a tyrosine-phosphorylated and ubiquitinated protein. Ymer is found to be phosphorylated at tyrosine 145 and 146 upon EGF stimulation, and lysine 129 of Ymer has been identified as a ubiquitination site. Ymer has two motifs interacting with the ubiquitin (MIU) domains that might function as a binding site for the ubiquitinated EGF receptor. Although Ymer and EGF receptors are associated in an EGF-dependent manner, their interaction is required not only for MIU domains but also for the tyrosine phosphorylation of Ymer. Phosphorylated Ymer is mainly located at the plasma membrane with EGF receptor and functions in its endocytosis and degradation. Furthermore, EGF-mediated secondary modifications of an activated-EGF receptor are inhibited by overexpressing Ymer in COS7 cells. Therefore, Ymer may have competitive effects on the activation of the EGF receptor. Our findings suggest that Ymer functions as a novel inhibitor for the down-regulation of the EGF receptor and plays a crucial role for regulating the amount of the EGF receptor on the cell surface membrane.

Opposing actions of the EGF family and opioids: heparin binding-epidermal growth factor (HB-EGF) protects mouse cerebellar neuroblasts against the antiproliferative effect of morphine

Neuregulins are a family of growth and differentiation factors that act through activation of cell-surface erbB receptor tyrosine kinases and have essential functions both during development and on the growth of cancer cells. One alternatively spliced neuregulin-1 form has a distinct heparin-binding immunoglobulin-like domain that enables it to adhere to heparan sulfate proteoglycans at key locations during development and substantially potentiates its activity. We examined the structural specificity needed for neuregulin-1-heparin interactions using a gel mobility shift assay together with an assay that measures the ability of specific oligosaccharides to block erbB receptor phosphorylation in L6 muscle cells. Whereas the N-sulfate group of heparin was most important, the 2-O-sulfate and 6-O-sulfate groups also contributed to neuregulin-1 binding in these two assays. Optimal binding to neuregulin-1 required eight or more heparin disaccharides; however, as few as two disaccharides were still able to bind neuregulin-1 to a lesser extent. The physiological importance of this specificity was shown both by chemical and siRNA treatment of cultured muscle cells. Pretreatment of muscle cells with chlorate that blocks all sulfation or with an siRNA that selectively blocks N-sulfation significantly reduced erbB receptor activation by neuregulin-1 but had no effect on the activity of neuregulin-1 that lacks the heparin-binding domain. These results suggest that the regulation of glycosaminoglycan sulfation is an important biological mechanism that can modulate both the localization and potentiation of neuregulin-1 signaling.

Dahl salt-sensitive (SS) rats fed a high salt diet exhibit increased blood pressure and renal damage. Epithelial sodium channels (ENaC) in the aldosterone-sensitive distal nephron (ASDN) are responsible for the fine tuning of Na + absorption in the kidney and are regulated by various stimuli including hormones and growth factors. Members of the epidermal growth factor (EGF) family are important for maintaining transepithelial Na + transport. A combination of electrophysiological, immunohistochemical, biochemical in vitro and chronic in vivo studies were used to provide mechanistic insights into ENaC regulation by EGF and how changes in this pathway may contribute to salt-induced hypertension in SS rats. It was found that the ENaC inhibitor benzamil, administered through the drinking water (15 mg/L), significantly reduced the MAP from 173.3±15.0 to 127.0±10.1 mm Hg. ENaC expression (as measured by Western blotting and immunohistochemistry) and activity (single channel analysis in freshly isolated split open ASDN) were significantly enhanced in SS rats fed a high salt diet compared to SS rats fed a low salt diet and consomic SS.13 BN rats fed a high salt chow. Servo-controlled measurements in SS rats also revealed increased ENaC expression in the uncontrolled kidneys compared to controlled left kidneys. As assessed by ELISA assay, EGF level in the kidney cortex of SS rats fed a high salt diet was lower compared to rats on a low salt diet. To directly evaluate the role of EGF in the development of hypertension and its effect on ENaC activity, EGF (10 μg/h/kg) was intravenously infused and blood pressure was continuously monitored. Chronic infusion of EGF decreased ENaC activity ( NP o ; from 1.5±0.2 to 0.4±0.1), prevented the development of hypertension (from 184.0±8.9 to 149.0±9.6 mm Hg) and attenuated renal glomerular and tubular damage. Thus, in controversy to existing dogma, we provide an evidence of a critical role of ENaC in the ASDN in the development of salt-sensitive hypertension. Furthermore, these findings indicate that EGF deficiency in the renal cortex contributes to salt-sensitive hypertension and that in vivo EGF infusion prevents development of hypertension and kidney damage via downregulation of ENaC.

Degradation of activated ERBB receptors is an important mechanism for signal attenuation. However, compared with epidermal growth factor (EGF) receptor, the ERBB2/ERBB3 signaling pair is considered to be attenuation-deficient. The ERBB2/ERBB3 ligands of the neuregulin family rely on an EGF-like domain for signaling and are generated from larger membrane-bound precursors. In contrast to EGF, which is processed to yield a 6-kDa peptide ligand, mature neuregulins retain a variety of segments N-terminal to the EGF-like domain. Here we evaluate the role of the N-terminal domain of neuregulin 1 in signaling and turnover of ERBB2/ERBB3. Our data suggest that whereas the EGF-like domain of neuregulin 1 is required and sufficient for the formation of active receptor heterodimers, the presence of the N-terminal Ig-like domain is required for efficient signal attenuation. This manifests itself for both ERBB2 and ERBB3 but is more pronounced and coupled directly to degradation for ERBB3. When stimulated with only the EGF-like domain, ERBB3 shows degradation rates comparable with constitutive turnover, but stimulation with full-length neuregulin 1 resulted in receptor degradation at rates that are comparable with activated EGF receptor. Most of the enhancement in down-regulation was maintained after replacing the Ig-like domain with a thioredoxin protein of comparable size but different amino acid composition, suggesting that the physical presence but not specific properties of the Ig-like domain are needed. This sequence-independent effect of the N-terminal domain correlates with an enhanced ability of full-size neuregulin 1 to disrupt higher order oligomers of the ERBB3 extracellular domains in vitro.

Cartilage matrix protein (CMP) exists as a disulfide-bonded homotrimer in the matrix of cartilage. Each monomer consists of two CMP-A domains that are separated by an epidermal growth factor-like domain. A heptad repeat-containing tail makes up the carboxyl-terminal domain of the protein. The secreted form of CMP contains 12 cysteine residues numbered C1 through C12. Two of these are in each of the CMP-A domains, six are in the epidermal growth factor-like domain, and two are in the heptad repeat-containing tail. Two major categories of mutant CMPs were generated to analyze the oligomerization process of CMP: a mini-CMP and a heptadless full-length CMP. The mini-CMP consists of the CMP-A2 domain and the heptad repeat-containing tail. In addition, a number of mutations affecting C9 through C12 were generated within the full-length, the mini-, and the heptad-less CMPs. The mutational analysis indicates that the heptad repeats are necessary for the initiation of CMP trimerization and that the two cysteines in the heptad repeat-containing tail are both necessary and sufficient to form intermolecular disulfide bonds in either full-length or mini-CMP. The two cysteines within a CMP-A domain form an intradomain disulfide bond.

Circular dichroism (CD) and Fourier transform infrared spectroscopic studies have shown that the secondary structure of transforming growth factor alpha (TGF-alpha) is very similar to that of epidermal growth factor (EGF). The infrared spectra revealed a minor difference between the two proteins, in particular in the beta-sheet structure. A large difference was observed with CD between the two proteins in the apparent conformation each adopts when the disulfide bonds are reduced. Reduced TGF-alpha showed a distinct alpha-helical conformation only at a high trifluoroethanol concentration, whereas reduced EGF assumed an alpha-helical conformation in the absence of trifluoroethanol. This indicates that these two proteins adopt different secondary structures in the absence of disulfide bonds, although they assume similar folding structures in their presence. These data suggest that the disulfide bonds to a large degree dictate the conformation of these two proteins. Additionally, differences in the dynamic behavior between EGF and TGF-alpha were also observed. Infrared experiments showed that the hydrogen-deuterium exchange rate is much higher for TGF-alpha than for EGF, indicating that TGF-alpha is a more flexible molecule. The rate of reduction of the disulfide bonds by dithiothreitol was also faster for TGF-alpha. Therefore, it can be concluded that although EGF and TGF-alpha have a similar overall conformation, TGF-alpha is a more flexible molecule than EGF.

The binding of factor IX to cultured bovine endothelial cells was characterized using isolated domains of bovine factor IX. An NH2-terminal fragment that consists of the gamma-carboxyglutamic acid (Gla) region linked to the two epidermal growth factor (EGF)-like domains bound to the endothelial cells with the same affinity as intact factor IX, indicating that the serine protease part of factor IX is not involved in binding. This fragment also inhibited the factor IXa beta'-induced clotting of plasma at a concentration that would suggest a competition for phospholipid binding sites. However, after proteolytic removal of the Gla region from the fragment, the two EGF-like domains inhibited clotting almost as effectively, suggesting a direct interaction between this part of the molecule and the cofactor, factor VIIIa. Using affinity-purified Fab fragments against the Gla region, the EGF-like domains, and the serine protease part, it was observed that the serine protease part of the molecule undergoes a large conformational change upon activation, whereas the Gla region and the EGF-like domains appear to be unaffected. All three classes of Fab fragments were equally efficient as inhibitors of the factor IXa beta'-induced clotting reaction. Part of factor Va and factor VIIIa have significant sequence homology to a lectin. We therefore investigated the effect on in vitro clotting of the recently identified unique disaccharide Xyl alpha 1-3Glc, that is O-linked to a serine residue in the NH2-terminal EGF-like domain of human factor IX (Hase, S., Nishimura, H., Kawabata, S.-I., Iwanaga, S., and Ikenaka, T. (1990) J. Biol. Chem. 265, 1858-1861). However, no effect on blood clotting was observed in the assay system used. Our results are compatible with a model in which the serine protease part provides the specificity of the binding of factor IXa to factor VIIIa-phospholipid, but that the EGF-like domain(s) also contributes to the interaction of the enzyme with its cofactor.

Members of the epidermal growth factor (EGF) family are frequently implicated in the injury response of the mammalian nervous system. Although this implication is supported by extensive molecular evidence, it is not underpinned by conclusive functional data. Recently, we found that expression of an EGF homolog from the pond snail Lymnaea stagnalis (L-EGF) is upregulated after axotomy in the adult CNS, suggesting a role for this molecule in the injury response of the CNS. In the present study we asked whether L-EGF can promote axonal regeneration of three types of identified neurons in organ-cultured CNS. Treatment with purified L-EGF substantially enhanced axonal regeneration of all three types of neurons, an effect inhibited by submicromolar doses of PD153035, a specific EGF receptor (EGFR) tyrosine kinase inhibitor. In addition, PD153035 and K252a, a nonspecific kinase inhibitor, also reduced the degree of axonal regeneration that occurs without L-EGF supplementation, indicating that L-EGF or other EGFR ligands synthesized in the CNS participate in the regenerative response. An intriguing aspect of these results is that axonal regeneration of different, intrinsically L-EGF responsive and unresponsive neurons occurred in a coordinated manner. This observation suggests that indirect in addition to direct actions contribute to the beneficial effect of L-EGF. In conclusion, we provide functional evidence that an EGF homolog can promote axonal regeneration, substantiating existing molecular evidence implicating the EGF family in peripheral nerve regeneration and emphasizes the therapeutic potential of these molecules.

BackgroundHeparin-binding epidermal growth factor-like growth factor (HB-EGF) belongs to the epidermal growth factor (EGF) family of growth factors. HB-EGF and its receptors, epidermal growth factor receptor (EGFR) and HER4, are expressed in the human corpus luteum. HB-EGF has been shown to regulate luteal function by preventing cell apoptosis. Steroidogenesis is the primary function of the human corpus luteum. Steroidogenic acute regulatory protein (StAR) plays a critical role in steroidogenesis. StAR expression and progesterone (P4) production in human granulosa-lutein (hGL) cells have been shown to be upregulated by a ligand of EGFR, amphiregulin. However, whether HB-EGF can achieve the same effects remains unknown.MethodsA steroidogenic human ovarian granulosa-like tumor cell line, KGN, and primary culture of hGL cells obtained from patients undergoing in vitro fertilization treatment were used as experimental models. The underlying molecular mechanisms mediating the effects of HB-EGF on StAR expression and P4 production were explored by a series of in vitro experiments.ResultsWestern blot showed that EGFR, HER2, and HER4 were expressed in both KGN and hGL cells. Treatment with HB-EGF for 24 h induced StAR expression but did not affect the expression of steroidogenesis-related enzymes, P450 side chain cleavage enzyme, 3β-hydroxysteroid dehydrogenase, and aromatase. Using pharmacological inhibitors and a siRNA-mediated knockdown approach, we showed that EGFR, HER4, but not HER2, were required for HB-EGF-stimulated StAR expression and P4 production. In addition, HB-EGF-induced upregulations of StAR expression and P4 production were mediated by the activation of the ERK1/2 signaling pathway.ConclusionThis study increases the understanding of the physiological role of HB-EGF in human luteal functions.7G2SU9-j2App9A69cYCW26Video

Epiregulin, a novel member of the epidermal growth factor family, is an autocrine growth factor in normal human keratinocytes

Traboulsi syndrome is an autosomal recessive hereditary disease associated with developmental defects, in particular of the ocular system. SNPs affecting the ASPH gene, which encodes for the 2-oxoglutarate (2OG)-dependent oxygenase aspartate/asparagine-β-hydroxylase (AspH), are associated with Traboulsi syndrome. AspH catalyzes hydroxylations of conserved aspartate/asparagine residues in epidermal growth factor–like domain (EGFD) proteins. We report studies on the clinically observed Traboulsi syndrome–associated R688Q, R735Q, and R735W AspH variants. The results reveal that pathogenic active site substitutions substantially reduce, though do not ablate, EGFD hydroxylase activity compared with wt AspH. They imply that efficient AspH-catalyzed EGFD hydroxylation is important during human development. Crystallographic studies reveal conservation of the overall AspH fold, but that the preferred conformations of 2OG in complex with the R735Q and R735W AspH variants differ from those with wt AspH. Screening of potential 2OG cosubstrate substitutes reveals that certain 2-oxoacids, including naturally present metabolites, manifest enhanced catalytic efficiency of Traboulsi syndrome–associated AspH variants compared with 2OG. The results thus provide proof of principle for a therapeutic strategy involving rescue of impaired activities of pathogenic active site AspH variants by use of 2-oxoacids, or 2-oxoacid precursors, other than 2OG.