Determining the binding affinities of prostate-specific antigen to lectins: SPR and microarray approaches.

Prostate cancer (PCa) is one of the most common newly diagnosed cancers among men and we focused on its traditional biomarker, prostate-specific antigen (PSA), using targeted glycomics-based strategies. The aberrant glycosylation pattern of PSA may serve as a valuable tool for improving PCa diagnosis including its early-stage. In this study, we evaluated the usability of two techniques, surface plasmon resonance and protein microarray assay, for the study and characterization of interactions of PSA (both free and complexed) with six lectins (SNA, ConA, RCA, AAL, WGA and MAA II). The information on the character of such interactions is important for the application of lectins as prospective bioreceptors for biomarker glycoprofiling in a follow-up biosensing assays. SPR as well as established bioanalytical techniques allowed determination of KD values of PSA-lectin interactions in a more reliable way than protein microarray. The protein microarray method did not allow accurate quantification of KD values. However, the features of a microarray approach, such as speed and costs, enabled the screening and estimation of the nature of lectin-glycan biomarker interaction in an effective and time-saving way. All of the tested lectins interacted with commercial PSA standard isolated from healthy persons, except MAA II which reacted only very weakly.

The expression of P-gp in leukemia cells is associated with cross-resistance to protein N-glycosylation inhibitor tunicamycin.

In P-gp-positive cell variants obtained from L1210 cells either by selection with vincristine (L1210/R) or by transfection with the human gene encoding P-gp (L1210/T), we have previously described cross-resistance to tunicamycin (TNM), a protein N-glycosylation inhibitor. Here we studied whether this cross-resistance also underlies P-gp-positive variants of human acute myeloid leukemia cells (AML) derived from SKM-1 and MOLM-13 cells (SKM-1/VCR, SKM-1/LEN, MOLM-13/VCR) by selection with vincristine (VCR) and lenalidomide (LEN). While SKM-1/LEN cells were P-gp positive, no P-gp was detected in MOLM-13/LEN cells. P-gp-positive cells could be repeatedly passaged in medium containing TNM. In contrast, more than 90% of P-gp-negative cells were entering and progressing through cell death mechanisms after the third passage in medium containing TNM. Combined apoptosis/necrosis cell death was detected in L1210 cells after exposure to TNM. Passaging of P-gp-negative AML cells in medium containing TNM induced preferentially apoptosis. Damage to P-gp-negative cells induced with TNM was associated with arrest in the G1 phase of the cell cycle. P-gp-positive leukemia cells differed from P-gp-negative cells in the composition of plasma membrane glycoproteins, which we monitored with the aid of different lectins. The application of TNM to cells induced additional changes in membrane-linked glycosides.

Screening for the best detergent for the isolation of placental membrane proteins.

Although membrane proteins (MPs) play crucial roles in physiological processes, information on them are insufficient, mostly due to their peculiar nature and surrounding which demand specific procedures for their extraction (using detergents) and analysis. A pallet of ten detergents and ß-cyclodextrin was employed to investigate their efficiency in extracting total placental MPs, glycoproteins and insulin-like growth factor receptors (IR/IGF1R/IGF2R). Regardless of detergent used, the identity of major extracted proteins was the same. Glycoproteins extracted with Triton X-100 contained the greatest variety and quantity of glycans recognised by fifteen lectins, pointing to this detergent as universal medium for the extraction of membrane glycoproteins. Glycoproteins extracted using Brij 35 exhibited weak interaction with only seven lectins and were differently recognised by lectins of the similar glycan specificity. Brij 35, Tween 20, saponin and digitonin selectively extracted IGF2R compared to other two receptors. Pilot experiments should be conducted in order to choose adequate detergent for the extraction of specific MP. To obtain preparations enriched in specific receptor of the insulin/IGF system sequential solubilisation of placental MPs can be proposed: to use Brij 35 to extract IGF2R and subject the insoluble remaining suspension to Triton X-114 in order to extract most of IGF1R with small amounts of IR.