Small Leucine Rich Proteoglycans as Novel Tumor Markers In Chronic Lymphocytic Leukemia

Abstract 694 Background: Small leucine rich proteoglycans (SLRPs) are a family of glycosylated proteins normally expressed in the extracelluar matrix (ECM) of collagen rich tissues. The biological role of the SLRPs is multifactoral, but mostly these proteins are secreted and bind to membrane receptors or ECM proteins affecting cell proliferation and cell migration. Some SLRPs (eg. Decorin, Lumican) have been reported to be expressed in cancer, but the expression as well as the glycosylation pattern differ. Microarray studies have revealed that the SLRP family member fibromodulin (FMOD) gene is overexpressed in chronic lymphocytic leukemia (CLL). We later reported the specific expression of FMOD in CLL and mantle cell lymphoma (MCL). FMOD is located on chromosome 1q32 adjacent to two other members of the SLRP family, proline/arginine-rich end leucine-rich repeat protein (PRELP) and opticin (OPTC). Aims: To analyse 1) the expression of PRELP and OPTC in CLL and other hematological malignancies. 2) the glycosylation pattern of PRELP and OPTC, and cellular localization of OPTC in CLL cells. Methods: PRELP: Gene expression was tested by RT-PCR and realtime-PCR. Protein expression was tested by western blot. Chemical deglycosylation was performed to characterize the glycosylation pattern of the PRELP protein. OPTC: Cell fractionation was performed using four different methods. Protein expression (molecular weight and cellular location) was tested by western blot. Chemical deglycosylation was performed to characterize the glycosylation pattern of the OPTC protein. Results: PRELP was expressed at the gene level (RT-PCR) in all CLL patients tested (n=30) and in 3/5 patients with mantle cell lymphoma, but not in normal leukocytes (n=10) or in other hematological malignancies (7 different types, n=35). The PRELP protein was detected in all CLL samples tested but not in leukocytes of healthy donors (western blot). Molecular analysis of the CLL PRELP protein revealed a unique unglycosylated 38 kDa core protein, with an intact signal peptide. This protein was not detected in serum that, in combination with the uncleaved signal peptide, suggests cellular retention. A “normal” OPTC protein (50 kDa) was detected in cell lysates of both CLL tumor cells (n=30) and normal leukocytes (n=10). However, the CLL cells also expressed a 37 kDa OPTC that was not detected in healthy controls. This 37 kDa OPTC was detected in all CLL samples and molecular analysis revealed a unique unglycosylated core protein that was located in the cell nucleus and endoplasmatic reticulum of the CLL cells. Conclusion: The unique expression of the SLRPs PRELP and OPTC in CLL (in addition to the previously reported FMOD) is unexpected and merits further studies since the specific expression of three closely related SLRP genes may indicate a role in the pathobiology of the disease. Disclosures: No relevant conflicts of interest to declare.

Carbohydrate Moieties of Microsporidian Polar Tube Proteins Are Targeted by Immunoglobulin G in Immunocompetent Individuals

ABSTRACT Microsporidia of the Encephalitozoon species are frequently found as opportunistic pathogens of immunocompromised patients, but very little is known about the prevalence and significance of Encephalitozoon infection in immunocompetent individuals. It was reported previously that 8% of Dutch blood donors and 5% of pregnant French women had an immunoglobulin G (IgG) immune response against specific organelles of Encephalitozoon intestinalis. These organelles, the so-called polar tube and anchoring disk, are used to penetrate membranes of host cells during infection. The unexpectedly high percentage of immunocompetent individuals with IgG against these organelles suggested that infection of humans with microsporidia might be more common than previously recognized. In the present study, we analyzed this anti-Encephalitozoon IgG response by using indirect immunofluorescence, Western blotting, two-dimensional gel electrophoresis, and chemical deglycosylation. Our results show that the antibody response is directed against the posttranslational carbohydrate modification of the major polar tube protein (polar tube protein 1) and carbohydrate moieties of proteins in the anchoring region of the polar tube of Encephalitozoon. In addition, the antibodies were found to decrease the infectivity of E. intestinalis in vitro. The significance and possible origin of these prevalent antibodies are discussed.

Partial deglycosylation of alpha subunit modifies sturgeon gonadotropin function.

Chemical deglycosylation (dg) of sturgeon Acipenser gueldenstaedti Br. (alphaGTH) resulted in the loss of 83% of its initial carbohydrate content. It altered also recombinant dg alphaGTH + betaGTH dimer molecule, reducing its immunoreactivity by 30%, and fully blocking the hormonal function. CD spectroscopy showed that deglycosylation led to changes in the secondary structure of dg alphaGTH and in the alpha-beta recombinant. The sugar moiety of sturgeon alphaGTH is suggested to play an important role in maintaining the biological function of the hormone dimer molecule.

Variable Carbohydrate Modifications to the Catalytic Chains of the RgpA and RgpB Proteases of Porphyromonas gingivalis W50

ABSTRACT Proteases of Porphyromonas gingivalis are considered to be important virulence determinants of this periodontal bacterium. Several biochemical isoforms of arginine-specific proteases are derived from rgpA and rgpB. HRgpA is a heterodimer composed of the catalytic α chain noncovalently associated with a β adhesin chain derived from the C terminus of the initial full-length translation product. The catalytic α chain is also present as a monomer (RgpA) either free in solution or associated with membranes.rgpB lacks the coding region for the adhesin domain present in rgpA and yields only monomeric forms (RgpB) which again may be soluble or membrane associated. In this study, the catalytic chains of this unusual group of enzymes are shown to be differentially modified by the posttranslational addition of carbohydrate. A monoclonal antibody (MAb 1B5) raised to the monomeric RgpA did not react with the corresponding recombinant RgpA α chain expressed inEscherichia coli but was immunoreactive with P. gingivalis lipopolysaccharide. MAb 1B5 also reacted with the membrane-associated forms of RgpA and RgpB but not with the heterodimeric HRgpA and the soluble form of RgpB. RgpA treated with denaturants was capable of binding to MAb 1B5 whereas treatment with periodate abolished this binding, suggesting the presence of carbohydrate residues within the epitope. Chemical deglycosylation abolished immunoreactivity with MAb 1B5 and caused a ∼30% reduction in the size of the membrane-associated enzymes. Monosaccharide analysis of HRgpA and RgpA demonstrated 2.1 and 14.4%, respectively, carbohydrate by weight of protein. Furthermore, distinct differences were detected in their monosaccharide compositions, indicating that these protease isoforms are modified not only to different extents but also with different sugars. The variable nature of these additions may have a significant effect on the structure, stability, and immune recognition of these protease glycoproteins.

Targeted Mutants of Cochliobolus carbonum Lacking the Two Major Extracellular Polygalacturonases

ABSTRACT The filamentous fungus Cochliobolus carbonum produces endo-α1,4-polygalacturonase (endoPG), exo-α1,4-polygalacturonase (exoPG), and pectin methylesterase when grown in culture on pectin. Residual activity in a pgn1 mutant (lacking endoPG) was due to exoPG activity, and the responsible protein has now been purified. After chemical deglycosylation, the molecular mass of the purified protein decreased from greater than 60 to 45 kDa. The gene that encodes exoPG, PGX1, was isolated with PCR primers based on peptide sequences from the protein. The product of PGX1, Pgx1p, has a predicted molecular mass of 48 kDa, 12 potential N-glycosylation sites, and 61% amino acid identity to an exoPG from the saprophytic fungus Aspergillus tubingensis. Strains of C. carbonum mutated in PGX1 were constructed by targeted gene disruption and by gene replacement. Growth of pgx1mutant strains on pectin was reduced by ca. 20%, and they were still pathogenic on maize. A double pgn1/pgx1 mutant strain was constructed by crossing. The double mutant grew as well as thepgx1 single mutant on pectin and was still pathogenic despite having less than 1% of total wild-type PG activity. Double mutants retained a small amount of PG activity with the same cation-exchange retention time as Pgn1p and also pectin methylesterase and a PG activity associated with the mycelium. Continued growth of thepgn1/pgx1 mutant on pectin could be due to one or more of these residual activities.

A novel keratan sulphate domain preferentially expressed on the large aggregating proteoglycan from human articular cartilage is recognized by the monoclonal antibody 3D12/H7

Monoclonal antibodies (mAbs) were prepared against aggrecan which has been isolated from human articular cartilage and purified by several chromatographic steps. One of these mAbs, the aggrecan-specific mAb 3D12/H7, was selected for further characterization. The data presented indicate that this mAb recognizes a novel domain of keratan sulphate chains from aggrecan: (1) immunochemical staining of aggrecan is abolished by treatment with keratanase/keratanase II, but not with keratanase or chondroitin sulphate lyase AC/ABC; (2) after chemical deglycosylation of aggrecan no staining of the core-protein was observed; (3) different immunochemical reactivity was observed against keratan sulphates from articular cartilage, intervertebral disc and cornea for the mAbs 3D12/H7 and 5D4. For further characterization of the epitope, reduced and 3H-labelled keratan sulphate chains were prepared. In an IEF–gel-shift assay it was shown that the 3H-labelled oligosaccharides obtained after keratanase digestion of reduced and 3H-labelled keratan sulphate chains were recognized by the mAb 3D12/H7. Thus it can be concluded that the mAb 3D12/H7 recognizes an epitope in the linkage region present in, at least some, keratan sulphate chains of the large aggregating proteoglycan from human articular cartilage. Moreover, this domain seems to be expressed preferentially on those keratan sulphate chains which occur in the chondroitin sulphate-rich region of aggrecan, since the antibody does not recognize the keratan sulphate-rich region obtained after combined chondroitinase AC/ABC and trypsin digestion of aggrecan.