Juan J. Calvete

Arg-Gly-Asp constrained within cyclic pentapoptides Strong and selective inhibitors of cell adhesion to vitronectin and laminin fragment P1

Cyclic Arg‐Gly‐Asp‐Phe‐Val peptides with either D‐Phe or D‐Val residues were 20‐ to more than 100‐fold better inhibitors of cell adhesion to vitronectin and/or laminin fragment P1 when compared to a linear variant or Gly‐Arg‐Gly‐Asp‐Ser. No or only little increase in inhibitory capacity was observed for fibronectin adhesion and for the binding or platelet receptor αIIbβ3 to fibrinogen. NMR studies of the two most active cyclic peptides showed for both an all‐trans conformation with a βII′ and γ turn. Subtle conformational differences, however, exist between both peptides and may contribute to selectivity or inhibition.

Cathepsin B efficiently activates the soluble and the tumor cell receptor-bound form of the proenzyme urokinase-type plasminogen activator (Pro-uPA).

Action of purified human cathepsin B on recombinant single-chain urokinase-type plasminogen activator (pro-uPA) generated enzymatically active two-chain uPA (HMW-uPA), which was indistinguishable by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot from plasmin-generated HMW-uPA and from elastase- or thrombin-generated inactive two-chain urokinase-type plasminogen activator. Preincubation of cathepsin B with E-64 (transepoxysuccinyl-L-leucylamino- (4-guanidino)butane, a potent inhibitor for cathepsin B) prior to the addition of pro-uPA prevented the activation of pro-uPA. The cleavage site within the cathepsin B-treated urokinase-type plasminogen activator (uPA) molecule, determined by N-terminal amino acid sequence analysis, is located between Lys158 and Ile159. Pro-uPA is cleaved by cathepsin B at the same peptide bond that is cleaved by plasmin or kallikrein. Binding of cathepsin B-activated pro-uPA to the uPA receptor on U937 cells did not differ from that of enzymatically inactive pro-uPA, indicating an intact receptor-binding region within the growth factor-like domain of the cathepsin B-treated uPA molecule. Not only soluble but also tumor cell receptor-bound pro-uPA could be efficiently cleaved by cathepsin B to generate enzymatically active two-chain uPA. Thus, cathepsin B can substitute for plasmin in the proteolytic activation of pro-uPA to enzymatically active HMW-uPA. In contrast, no significant activation of pro-uPA by cathepsin D was observed. As tumor cells may produce both pro-uPA and cathepsin B, implications for the activation of tumor cell-derived pro-uPA by cellular proteases may be considered.

Crystal structure of the complex formed by the membrane type 1-matrix metalloproteinase with the tissue inhibitor of metalloproteinases-2, the soluble progelatinase A receptor

The proteolytic activity of matrix metalloproteinases (MMPs) towards extracellular matrix components is held in check by the tissue inhibitors of metalloproteinases (TIMPs). The binary complex of TIMP‐2 and membrane‐type‐1 MMP (MT1‐MMP) forms a cell surface located ‘receptor’ involved in pro‐MMP‐2 activation. We have solved the 2.75 Å crystal structure of the complex between the catalytic domain of human MT1‐MMP (cdMT1‐MMP) and bovine TIMP‐2. In comparison with our previously determined MMP‐3–TIMP‐1 complex, both proteins are considerably tilted to one another and show new features. CdMT1‐MMP, apart from exhibiting the classical MMP fold, displays two large insertions remote from the active‐site cleft that might be important for interaction with macromolecular substrates. The TIMP‐2 polypeptide chain, as in TIMP‐1, folds into a continuous wedge; the A‐B edge loop is much more elongated and tilted, however, wrapping around the S‐loop and the β‐sheet rim of the MT1‐MMP. In addition, both C‐terminal edge loops make more interactions with the target enzyme. The C‐terminal acidic tail of TIMP‐2 is disordered but might adopt a defined structure upon binding to pro‐MMP‐2; the Ser2 side‐chain of TIMP‐2 extends into the voluminous S1′ specificity pocket of cdMT1‐MMP, with its Oγ pointing towards the carboxylate of the catalytic Glu240. The lower affinity of TIMP‐1 for MT1‐MMP compared with TIMP‐2 might be explained by a reduced number of favourable interactions.

Spermadhesins: A new protein family. Facts, hypotheses and perspectives

Summary Spermadhesins are a novel family of secretory proteins expressed in the male genital tract of pig, horse and bull. They are major products of the seminal plasma and have been found to be peripherally associated to the sperm surface. The structure and function of spermadhesins have been thoroughly investigated in the pig, which exhibits the greatest diversity of members: AWN, AQN‐1, AQN‐2, PSP‐I and PSP‐II and its glycosylated isoforms. They are multifunctional proteins showing a range of ligand‐binding abilities, e.g. carbohydrates, sulfated glycosamino‐glycans, phospholipids and protease inhibitors, suggesting that they may be involved in different steps of fertilization. Isolated porcine spermadhesins bind the zona pellucida glycoproteins in a cation‐dependent manner with a Kd in a low micromolar range, and AWN, AQN‐1 and AQN‐3 display similar binding affinity for glyoproteins containing Galβ(1–3)‐GalNAc and Galβ(1–4)‐GlcNAc sequences in O‐linked and N‐linked oligosaccharides, respectively. During sperm passage through the epididymis AQN‐3 and AWN have been shown to bind tightly to the sperm surface by interaction with the phospholipids of the membrane bilayer. At ejaculation the spermadhesins form a protective coat around the sensitive acrosomal region of the sperm head, thus possibly preventing premature acrosome reaction. During in vitro capacitation most of these aggregated sperm adhesins are lost, with the exception of phospholipid‐bound spermadhesins. AWN and AQN‐3 may now serve as a primary receptor for the oocyte zona pellucida, thus contributing to initial binding and recognition between sperm and egg.

Effective activation of the proenzyme form of the urokinase‐type plasminogen activator (pro‐uPA) by the cysteine protease cathepsin L

Increased levels of both the cysteine protease, cathepsin L, and the serine protease, uPA (urokinase‐type plasminogen activator), are present in solid tumors and are correlated with malignancy. uPA is released by tumor cells as an inactive single‐chain proenzyme (pro‐uPA) which has to be activated by proteolytic cleavage. We analyzed in detail the action of the cysteine protease, cathepsin L, on recombinant human pro‐uPA. Enzymatic assays, SDS‐PAGE and Western blot analysis revealed that cathepsin L is a potent activator of pro‐uPA. As determined by N‐terminal amino acid sequence analysis, activation of pro‐uPA by cathepsin L is achieved by cleavage or the Lys158‐lle159 peptide bond, a common activation site of serine proteases such as plasmin and kallikrein. Similar to cathepsin B (Kobayashi et al., J. Biol. Chem. (1991) 266, 5147‐5152) cleavage of pro‐uPA by cathepsin L was most effective at acidic pH (molar ratio of cathepsin L to pro‐uPA of 1:2,000). Nevertheless, even at pH 7.0, pro‐uPA was activated by cathepsin L, although a 10‐fold higher concentration of cathepsin L was required. As tumor cells may produce both pro‐uPA and cathepsin L, implications for the activation of tumor cell‐derived pro‐uPA by cathepsin L may be considered. Different pathways activation of pro‐uPA in tumor tissues may coexist: (i) autocatalytic intrinsic activation of pro‐uPA; (ii) activation by serine proteases (plasmin, kallikrein. Factor XIIa); and (iii) activation by cysteine proteases (cathepsin B and L).

Isolation and characterization of heparin- and phosphorylcholine-binding proteins of boar and stallion seminal plasma. Primary structure of porcine pB1

In the bovine, seminal plasma heparin‐binding proteins bind to sperm lipids containing the phosphorylcholine group and mediate the capacitating effects of heparin‐like glycosaminoglycans during sperm residence in the female genital tract. We report the characterization of heparin‐ and phosphorylcholine‐binding proteins of stallion and boar seminal plasma. orse eminal lasma proteins HSP‐1 and HSP‐2, and boar protein pB1, belong to the same family as the bull heparin‐ and phosphorylcholine‐binding proteins BSP‐A1/2, BSP‐A3, and BSP‐30K. We have determined the amino acid sequence and posttranslational modifications of boar glycoprotein pB1. It contains 105 amino acids arranged into a mosaic structure consisting of a N‐terminal 18‐residue O‐glycosylated polypeptide followed by two tandemly organized 40–45‐residue fibronectin type II domains. pB1 displays 60–65% amino acid sequence similarity with its equine and bovine homologues. However, in their respective seminal plasmas, the BSP and the HSP proteins associate into 90–150‐kDa oligomeric complexes, whereas pB1 forms a 35–40‐kDa complex with spermadhesin AQN‐1. In addition, pB1 appears to be identical to the recently described leukocyte adhesion regulator of porcine seminal fluid pAIF‐1. Our results tie in with the hypothesis that homologous proteins from different mammalian species may display distinct biological activities, which may be related to species‐specific aspects of sperm physiology.

Influence of Porcine Spermadhesins on the Susceptibility of Boar Spermatozoa to High Dilution

Abstract The effect of heparin-binding and non-heparin-binding spermadhesins on the viability, motility, and mitochondrial activity of boar spermatozoa at the high dilution (300 000 sperm/ml) to which sperm are exposed during the process of sex sorting by flow cytometry was investigated. Incubation of spermatozoa with heparin-binding spermadhesins caused a time- and dose-dependent decrease in the percentage of functional spermatozoa. The percentage of viable spermatozoa incubated at 38°C with heparin-binding spermadhesins diluted in PBS (1 mg/ml) dropped from 75% (0.5 h) to 4% (5 h), whereas the percentage of viable spermatozoa incubated in PBS without proteins (control) decreased from 85% (0.5 h) to 19% (5 h). Addition of non-heparin-binding PSP-I/PSP-II spermadhesin to the PBS resulted in a concentration-dependent increment of the percentage of viable cells (65% after 5-h incubation), with maximum effect at 1.5 mg/ml. The heparin-binding spermadhesins totally suppressed sperm motility and mitochondrial activity after 5 h of incubation. The same parameters of sperm incubated in the presence of 1.5 mg/ml of PSP-I/PSP-II were 50% and 58%, respectively, and the percentages of control sperm displaying motility and mitochondrial activity were 21% and 26%, respectively. Moreover, the viability, motility, and mitochondrial activity all decreased on incubation of spermatozoa with mixtures of PSP-I/PSP-II and heparin-binding spermadhesins as the concentration of the latter increased. We conclude that PSP-I/PSP-II and the heparin-binding spermadhesins exert antagonistic effects on the functionality of highly diluted boar spermatozoa. The finding that PSP-I/PSP-II contributes to maintaining sperm with high viability, motility, and mitochondrial activity for at least 5 h at physiological temperature points to its potential use as an additive for sperm preservation, specifically of highly diluted, flow-sorted spermatozoa for sex preselection.

Quantitation of boar spermadhesins in accessory sex gland fluids and on the surface of epididymal, ejaculated and capacitated spermatozoa

Spermadhesins are multifunctional proteins involved in boar sperm capacitation and gamete recognition. Using anti-AWN antibodies, we have followed the fate of spermadhesin AWN along the maturation and capacitation stages of boar spermatozoa. In addition, the amount of spermadhesins AQN-1, AQN-2, and AQN-3 relative to that of AWN was determined by amino acid analysis after reverse-phase HPLC isolation. Our data show that AWN-1 is the only spermadhesin on the surface of epididymal sperm and that a large amount of AQN-1, AQN-2, AQN-3, AWN-1 and AWN-2 become coated on ejaculated spermatozoa. The number of spermadhesin molecules on ejaculated sperm (12-60 x 10(6)/spermatozoa) is sufficient to form a many-molecules-thick coat over the sperm head. However, 50-75% of the AQN-1, AQN-2, and AQN-3 population, and around 90% of coated AWN (1 + 2) are released from ejaculated sperm during capacitation. This indicates that a large subpopulation of each boar spermadhesin is loosely associated to the sperm surface and may function as decapacitation factors. The remaining spermadhesin molecules, which are tightly bound to the sperm heads surface may play a role as either positive capacitation factors and/or in gamete recognition and binding.

The complete primary structure of the spermadhesin AWN, a zona pellucida-binding protein isolated from boar spermatozoa

AWN is a boar protein which originates in secretions of the male accessory glands and which becomes sperm surface‐associated upon ejaculation. It is one of the components thought to mediate sperm adhesion to the eggs zona pellucida through a carbohydrate‐recognition mechanism. AWN may, thus, participate in the initial events of fertilization in the pig. In this report we describe its complete primary structure by combination of protein‐chemical and mass spectrometric methods. AWN exists as two isoforms, AWN‐1 and AWN‐2, which differ in that AWN‐2 is N‐terminally acetylated. The amino acid sequence of AWN contains 133 amino acid residues and two disulphide bridges between nearest‐neighbour cysteine residues. Analysis of the amino acid sequence of the AWN proteins showed significant similarity only to AQN‐1 and AQN‐3, two other boar spermadhesins.

Biophysical characterization of the interaction of bovine seminal plasma protein PDC-109 with phospholipid vesicles

Abstract PDC-109 is the major protein of bovine seminal plasma. It binds to the bovine sperm surface at ejaculation and modulates sperm capacitation. PDC-109 displays phosphorylcholine- and heparin-binding activities which are thought to account for its sperm surface coating and glycosaminoglycan-induced sperm capacitating activities, respectively. We have characterized the interaction of isolated PDC-109 with membranes of phospholipid vesicles using a biophysical approach. Our results show that PDC-109 interacts not only with the solvent-exposed phosphorylcholine head group but also with the hydrophobic core of liposomes. Binding of PDC-109 to membranes is a very rapid, biphasic process with half times of less than one second. Maximal binding of PDC-109 to small unilamellar vesicles was achieved with a stoichiometric ratio of 10–11 phosphatidylcholine molecules/PDC-109 molecule. Incorporation of phosphatidylethanolamine or phosphatidylserine into phosphatidylcholine vesicles reduced the binding of PDC-109, suggesting that both the density of phosphorylcholine groups and the surface charge determine the interaction of the seminal plasma protein with the surface of the membrane. Electron spin resonance measurements showed that binding of PDC-109 to phosphatidylcholine vesicles caused a rigidification of the membrane. The relevance of the data for describing the role of PDC-109 in the modulation of sperm capacitation is discussed.