Xinli Lin

Cloning and Characterization of cDNA Encoding Cardosin A, an RGD-containing Plant Aspartic Proteinase

Cardosin A is an abundant aspartic proteinase from pistils of Cynara cardunculus L. whose milk-clotting activity has been exploited for the manufacture of cheese. Here we report the cloning and characterization of cardosin A cDNA. The deduced amino acid sequence contains the conserved features of plant aspartic proteinases, including the plant-specific insertion (PSI), and revealed the presence of an Arg-Gly-Asp (RGD) motif, which is known to function in cell surface receptor binding by extracellular proteins. Cardosin A mRNA was detected predominantly in young flower buds but not in mature or senescent pistils, suggesting that its expression is likely to be developmentally regulated. Procardosin A, the single chain precursor, was found associated with microsomal membranes of flower buds, whereas the active two-chain enzyme generated upon removal of PSI is soluble. This result implies a role for PSI in promoting the association of plant aspartic proteinase precursors to cell membranes. To get further insights about cardosin A, the functional relevance of the RGD motif was also investigated. A 100-kDa protein that interacts specifically with the RGD sequence was isolated from octyl glucoside pollen extracts by affinity chromatography on cardosin A-Sepharose. This result suggests that the 100-kDa protein is a cardosin A receptor and indicates that the interaction between these two proteins is apparently mediated through RGD recognition. It is possible therefore that cardosin A may have a role in adhesion-mediated proteolytic mechanisms involved in pollen recognition and growth.

Construction of retroviral producer cells from adenoviral and retroviral vectors

A combination of adenoviral and retroviral vectors was used to construct second generation packaging cells that deliver marker genes to target cells. A vector based upon Moloney murine leukemia virus (MoMLV) was used to deliver marker genes, and an adenovirus-based delivery system was used to deliver MoMLV structural genes (gag pol and env) to cultured cells. The procedure transformed the cells into new retroviral producer cells, which generate replication-incompetent retroviral particles in the culture supernatant for transferring marker genes to target cells. The titer of the retroviral-containing supernatant generated from the second generation producer cells reached above 105 c.f.u./ml, which is comparable to the MoMLV-based producer cell lines currently used in human gene therapy trials. These observations suggest that this new gene transfer scheme is technically feasible. The vector and procedures may be adapted for experimental human gene therapy in which the new producer cells are transplanted into patients for continuous gene transfer.

[12] Relationships of human immunodeficiency virus protease with eukaryotic aspartic proteases

Publisher Summary This chapter describes the methods used in the comparative studies of the evolutionary relationships and catalytic mechanism of HIV-1 protease and representative eukaryotic aspartic proteases. The evidence for the evolutionary relationship of eukaryotic and retroviral aspartic proteases originates from the observation that the single-chain eukaryotic aspartic proteases contain internal twofold similarity in secondary and tertiary structures that was postulated to be derived from gene duplication and fusion in the evolution of this group of enzymes. Mature HIV-1 protease is a homodimer with a monomer size of 99 amino acids. Its three-dimensional structure retains the important core-β structure of eukaryotic aspartic proteases, while the conformation of the active site, including the aspartic acids, is virtually identical to those of the eukaryotic enzymes. In both HIV and eukaryotic enzymes, a water molecule is found to be hydrogen bonded between the two active site aspartic carboxyls. Like the eukaryotic enzyme, HIV protease has a substrate-binding cleft that contains at least eight substrate side chain-binding pockets. These similarities suggest that HIV protease and the eukaryotic aspartic proteases are related in evolution and are similar in their catalytic mechanisms. Therefore, comparative studies of these enzymes may provide insights into their structure–function relationships.

Structural and Functional Aspects of Cardosins

Cardosins are aspartic proteinases of the flowers of Cynara cardunculus L. These flowers have economical relevance in Portugal since they are traditionally used in the manufacture of highly appreciated ewe cheeses such as Serra, Azeitao and Serpa. Although the milk-clotting activity of the cardoon has been exploited for centuries, the biochemistry of the process was relatively unknown until some years ago when we and other laboratories started to study both basic and applied aspects of the cardoon preparation [1–7]. In a first stage it was demonstrated that the milk-clotting activity is due to the presence of aspartic proteinases which cleave the peptide bond Phe 105–Met 106 of k-casein [1,3], a bond also cleaved by other milk-clotting enzymes used for cheese making [8]. Cleavage of this bond is known to induce destabilization of the casein micelle and subsequent formation of a clot, [9], and thus it is possible that the milk-clotting process induced by cardoon proteinases occurs in a similar way. However, the organoleptic properties of the products obtained with the flower of cardoon are clearly different from those of cheeses made from the same milk with chymosin or microbial rennets [10], stressing the unique characteristics of the cardoon enzymes.

Substrate Specificity Study of Recombinant Rhizopus Chinensis Aspartic Proteinase

Rhizopuspepsin, a model aspartic proteinase from the fungus Rhizopus chinensis, has recently been cloned and expressed by Chen et al. (1991). High resolution crystallographic analysis of rhizopuspepsin and complexes with active site ligands has been reported by Davies’ group (Parris et al., this volume). Our initial characterization of the substrate specificity of the active site is described in this report. This study will enable future comparisons between kinetic and crystallographic data from other aspartic proteinases as well as for use in planning and analyzing site-directed mutagenesis studies.

Engineering aspartic proteases to probe structure and function relationships

Recently, protein engineering has been used to interconvert homodimeric and homologous single-chain aspartic proteases, with some success. The independent folding of the domains of these proteases has also permitted the engineering of domain-rearranged protease zymogens and the use of individual domains as probes for structural denaturation. In addition, site-directed mutagenesis has provided insights into the catalytic mechanism and specificity of this family of proteases.

Thermopsin, A Thermostable Acid Protease from Sulfolobus Acidocaldarius

Most of the well-studied aspartic proteases, including those derived from yeast, fungi, plants and animal sources, are stable in temperatures up to about 50° to 60°C. Aspartic proteases which can function at high temperature in the range of 80° to 100°C have not been reported so far. We searched for thermostable acid proteases in the thermoacidophilic archaebacteria, Sulfolobus acidocaldarius, Sulfolobus solfataricus, and Thermoplasma acidophilum, because these organisms grow best in acidic media in a pH near 2 and at temperature of 80°C. Using a highly sensitive radioassay (Lin et al, 1989), we found proteolytic activities in the cultures of all three bacteria. The highest activity was found in Sulfolobus acidocaldarius. This protease was named thermopsin.

Primary substrate specificities of secreted aspartic proteases of Candida albicans.

Candidiasis is an opportunistic infection of a pathogenic yeast of the genus Candida. These otherwise benign commensals become opportunistically invasive in response to impaired host defense mechanisms. Prominent among putative virulence factors are the secreted aspartic proteases, or Saps (Goldman et al., 1995; Ruchel et al., 1992; Cutler, 1991) which are implicated by biochemical, genetic, and immunochemical evidence. Multiple genes encoding Saps have been identified in the genome of clinical isolates of Candida species (Monod et al., 1994). In the most virulent species, C. albicans, seven genes have been cloned (SAP1—SAP7; Monod et al., 1994). The induction of SAP gene expression is strictly regulated in accord with changes in cell phenotype and morphology (Hube et al., 1994), implying that Candida albicans may require stage-specific proteases for its life cycle (Odds, 1994). Of these enzymes, SAP6 was found to be expressed exclusively in the virulent form of these cells, implying a direct invasive function (Hube et al., 1994; White and Agabian, 1995).