Carotenoids from Haloarchaea: Extraction, Fractionation, and Characterization.

Carotenoids are bioactive molecules known to promote human health. Many extreme halophilic archaea synthesize carotenoids, mainly represented by C50 bacterioruberin (BR) and its derivatives. BR has a potent antioxidant capacity, even higher than that of ß-carotene, thus, there is an increasing interest to advance the study of its biological properties as well as to extend its current applications. Here, we describe a procedure to extract and characterize carotenoids (enriched in BR) from haloarchaea using a "hyperpigmented" genetically modified strain of Haloferax volcanii.

Proteolytic Activity Assays in Haloarchaea.

Extreme halophilic archaea (haloarchaea) have adapted their physiology and biomolecules to thrive in saline environments (>2 M NaCl). Many haloarchaea produce extracellular hydrolases (including proteases) with potential biotechnological applications, which require unusual high salt concentrations to attain their function and maintain their stability. These conditions restrict many of the standard methods used to study these enzymes such as activity determination and/or protein purification. Here, we describe basic protocols to detect and measure extracellular proteolytic activity in haloarchaea including casein hydrolysis on agar plates, quantitative proteolytic activity determination by the azocasein assay and gelatin zymography in presence of the compatible solute glycine-betaine.

A rhomboid protease gene deletion affects a novel oligosaccharide N-linked to the S-layer glycoprotein of Haloferax volcanii.

Rhomboid proteases occur in all domains of life; however, their physiological role is not completely understood, and nothing is known of the biology of these enzymes in Archaea. One of the two rhomboid homologs of Haloferax volcanii (RhoII) is fused to a zinc finger domain. Chromosomal deletion of rhoII was successful, indicating that this gene is not essential for this organism; however, the mutant strain (MIG1) showed reduced motility and increased sensitivity to novobiocin. Membrane preparations of MIG1 were enriched in two glycoproteins, identified as the S-layer glycoprotein and an ABC transporter component. The H. volcanii S-layer glycoprotein has been extensively used as a model to study haloarchaeal protein N-glycosylation. HPLC analysis of oligosaccharides released from the S-layer glycoprotein after PNGase treatment revealed that MIG1 was enriched in species with lower retention times than those derived from the parent strain. Mass spectrometry analysis showed that the wild type glycoprotein released a novel oligosaccharide species corresponding to GlcNAc-GlcNAc(Hex)2-(SQ-Hex)6 in contrast to the mutant protein, which contained the shorter form GlcNAc2(Hex)2-SQ-Hex-SQ. A glycoproteomics approach of the wild type glycopeptide fraction revealed Asn-732 peptide fragments linked to the sulfoquinovose-containing oligosaccharide. This work describes a novel N-linked oligosaccharide containing a repeating SQ-Hex unit bound to Asn-732 of the H. volcanii S-layer glycoprotein, a position that had not been reported as glycosylated. Furthermore, this study provides the first insight on the biological role of rhomboid proteases in Archaea, suggesting a link between protein glycosylation and this protease family.

Partial purification and characterization of a trypsin-like serine protease from bovine sperm.

Sperm proteolytic activities are relevant in the enzymatic mechanism of fertilization. Several authors have suggested the presence of serine proteases other than acrosin in mice and human spermatozoa. In this work we describe the characterization of a partially purified bovine sperm serine protease BSp66 and its dimmer, BSp120. Partial purification of the monomer was performed from fresh spermatozoa, while the dimer form of the protease was obtained from cryopreserved spermatozoa. The Mr of BSp120 and BSp66 estimated by zymography and gel filtration chromatography were 120 and 66 kDa, respectively. They were positively stained by Schiff-PAS reagent for glycoproteins and they both digested synthetic peptides with basic amino acids in the P1 site. Polyclonal antibodies against acrosin or proacrosin did not cross-react neither with BSp120, nor BSp66. In addition, antibodies raised in our laboratory against BSp120 and BSp66 did not recognize acrosin or proacrosin suggesting that they are not antigenically related proteins. Also, no cross- reactivity was detected with proteins in the range of 120-66 kDa when antibodies against the proteasome were used. The cellular localization of this protease by optical immunocytochemistry using specific antibodies revealed a positive signal in the apical portion of the sperm head suggesting acrosomal or membrane localization. The evidences presented here characterize BSp66 as a trypsin-like serine protease, a putative new member of this highly redundant proteolytic system of the sperm acrosome.

Low temperature-induced dimerization of the bovine sperm serine protease, BSp66.

BSp120 and BSp66 are trypsin-like serine proteases from bovine spermatozoa. The former is active in cryopreserved sperm samples while the latter shows proteolytic activity in recently obtained fresh sperm. Both proteases are immunologically related and co-localize in the apical portion of the sperm head. In Western blots with specific antibodies, sperm samples incubated with reducing agents showed a decrease in the amount of BSp120, while BSp66 was detected with both anti-BSp120 and anti-BSp66 antibodies. BSp120 was evident in frozen intact spermatozoa after 60 days of semen cryopreservation and the kinetic of appearance of this protein was coincident with the decrease in the amount of BSp66. Identical results were obtained by freezing sperm extracts from fresh semen at -20 degrees C. Our results suggest that BSp120 results from disulfide bond-dimerization of BSp66 and that this process may be induced by temperatures below zero in both intact spermatozoa and in sperm extracts.

Ubiquitin-like proteins in halobacteria.

Within our studies of protein degradation, the presence of ubiquitinylated proteins in haloalkaliphilic archaea was investigated. We found that Natronococcus occultus proteins that react with antibodies raised against ubiquitin appear in different growth phases, particularly in the initial and exponential ones. The expression of these proteins is increased when the cells are either treated with puromycin or starved for nutrients. Dot blot analysis of cell extracts with antibody against ubiquitin shows the presence of either ubiquitinylated or ubiquitin-like proteins not only in Natronococcus occultus, but also in various genera of halobacteria.