A sensitive assay to measure biomarker glycosylation demonstrates increased fucosylation of prostate specific antigen (PSA) in patients with prostate cancer compared with benign prostatic hyperplasia
Introduction
Prostate specific antigen (PSA) has been used for the diagnosis of prostate cancer (PCa) and monitoring patient response to treatment for approximately 20 years. PSA is a serine protease belonging to the kallikrein family, produced in the epithelium of the periurethral glands and functions to liquefy seminal gel [1]. The enzyme is a single chain glycoprotein with one N-linked glycosylation site at Asn-45; the molecular mass is 26,079 Da and the carbohydrate moiety adds approximately 2300 Da further to the overall weight, therefore, free PSA (fPSA) is approximately 28,400 Da [2].
One of the inherent problems of PSA use in the diagnostic setting is the poor specificity for PCa as patients with benign prostatic hyperplasia (BPH) may exhibit serum levels of PSA of 4 ng/ml or higher. Various approaches have been used to improve the selectivity and sensitivity of PSA for PCa, for example, PSA density [3] and PSA velocity [4]. PSA is found in two predominant forms in the serum either as fPSA or complexed with α1-chymotrypsin and α2-macroglobulin [5]. PCa patients have less fPSA than patients with BPH [6] and the ratio of fPSA to total PSA (tPSA) is used for the diagnosis of PCa but is not without problems, as the levels of fPSA vary with age [7].
The modification of proteins by glycosylation occurs in approximately half of all serum proteins and changes in glycosylation have been found to accompany both tumour formation and aggressive clinical behaviour [8]. Aberrant glycosylation of proteins in PCa tissue has been observed compared with BPH and normal tissue [9], [10], [11], [12]. Similarly, changes in the glycans on PSA have been observed in PCa compared with BPH but studies in this area have either used very few samples with extremely high PSA levels or technology only available in specialised glycobiology/glycoproteomic laboratories [13], [14], [15], [16].
We hypothesised that alterations in the tissue fucosylation of PCa compared with BPH [12] would be reflected in changes in the glycosylation of PSA and that this might add an element of specificity to the PSA test. The lectin from Ulex europaeus (UEA-1; specific for α1,2 linked fucose) was used and showed an elevation in the fucosylation of immuno-purified PSA from PCa serum samples compared with BPH. The observations were validated using a separate set of 26 serum samples in a simple enzyme-linked immunosorbent lectin assay (ELLA) developed in-house. The ELLA system – when used to study fPSA fucosylation – showed 92% specificity and 69% sensitivity for PCa over BPH, this compared with 70% specificity and 56% sensitivity for fPSA alone, when the threshold value was taken of 25% of the tPSA levels.
Section snippets
Subjects and sample collection
All patients had pathologically confirmed BPH or PCa and gifted a blood sample to the study, the patient details are given in Table 1. The study was approved by the Local Ethics Committee at the University College London Hospital Trust. 10 ml of blood was collected pre-operatively from each patient after informed consent. A sample of the blood was sent for routine PSA testing and the remainder was allowed to clot; centrifuged at 1500 g for 5 min, the supernatant was transferred to a new tube and
UEA-1 binding to immuno-purified PSA
Serum PSA from patients with PCa and BPH was separated by SDS-PAGE and probed with UEA-1, or anti-PSA antibody, Fig. 1. Several bands of MW lower than fPSA were observed; these most probably represent clipped forms of PSA. The higher MW bands (between 50 kDa and 100 kDa) are likely to be PSA complexed with serum proteins. Fucosylation of affinity purified PSA from serum of patients with PCa and BPH was compared by assessing the ratio of UEA-1 to PSA binding (Fig. 1) and was observed to be
Discussion
In this study an ELLA was developed in which the lectin UEA-1 was used to assess the glycosylation of the protein biomarker PSA. Only 50 μl of serum sample from patients with PSA in the “grey zone” range of 4–10 ng/ml was used in the assay.
The results showed an increase in the fucosylation of serum PSA in the PCa patient samples. An important next development for this field will be the availability of a recombinant form of PSA with “normal” and “cancer” glycoforms, these will find use as
Acknowledgements
This study was supported by a grant from Against Breast Cancer (registered charity 1121258) for AJ. Conflicts of interest: MD and AL are inventors on a British Patent (number 2379444) that is based on the observations described in this manuscript.
References (25)
- et al.
cDNA coding for the entire human prostate specific antigen shows high homologies to the human tissue kallikrein genes
Biochem Biophys Res Commun
(1989) - et al.
Prostate specific antigen density: a means of distinguishing benign prostatic hypertrophy and prostate cancer
J Urol.
(1992) - et al.
Serum prostate specific antigen complexed to alpha 1-antichymotrypsin as an indicator of prostate cancer
J Urol
(1993) - et al.
The value of screening tests in the detection of prostate cancer. Part II: retrospective analysis of free/total prostate-specific analysis ratio, age-specific reference ranges, and PSA density
Urology
(1995) - et al.
Focused differential glycan analysis with the platform antibody-assisted lectin profiling for glycan-related biomarker validation
Mol Cell Proteomics
(2009) - et al.
Enzyme linked lectin assay (ELLA) for direct analysis of transferrin sialylation in serum samples
Clin Biochem
(2007) - et al.
Glycosylation of urinary prostate-specific antigen in benign hyperplasia and cancer: assessment by lectin binding patterns
Clin Biochem
(2005) - et al.
Lectin and serum-PSA interaction as a screening test for prostate cancer
Clin Biochem
(2003) - et al.
Molecular mass and carbohydrate structure of prostate specific antigen: studies for establishment of an international PSA standard
Prostate
(1995) - et al.
Longitudinal evaluation of prostate-specific antigen levels in men with and without prostate disease
JAMA
(1992)
Multicenter comparison of the diagnostic performance of free prostate-specific antigen
Urology
Harnessing changes in cellular glycosylation in new cancer treatment strategies
Curr Cancer Drug Targets
Cited by (62)
Lectins applied to diagnosis and treatment of prostate cancer and benign hyperplasia: A review
2021, International Journal of Biological MacromoleculesDetection of bladder cancer with aberrantly fucosylated ITGA3
2021, Analytical BiochemistryA highly sensitive aptasensor for the detection of prostate specific antigen based on dumbbell hybridization chain reaction
2021, Sensors and Actuators, B: ChemicalGlycosylation markers in cancer
2019, Advances in Clinical ChemistryCitation Excerpt :In cholangiocarcinoma, serum MUC5AC was detected using an antibody-lectin sandwich ELLA containing 22C5, a MUC5AC specific monoclonal antibody, combined with soybean agglutinin (SBA) [44]. Serum fucosylated PSA, a marker for prostate cancer, was measured using an antibody-lectin sandwich ELLA containing a specific monoclonal antibody in combination with Ulex europaeus agglutinin-1 (UEA-1) [81]. In many studies, some lectins were selectively reactive to cancer cells.
An endoglycosidase-assisted LC-MS/MS-based strategy for the analysis of site-specific core-fucosylation of low-concentrated glycoproteins in human serum using prostate-specific antigen (PSA) as example
2018, Clinica Chimica ActaCitation Excerpt :These types of assays are based on a similar principle to the common enzyme-linked immunosorbent assay (ELISA) technique, in which the capture or detection antibody or both are replaced by a lectin. Approaches involving antibody-based capture of low-concentrated glycoproteins and subsequent detection of their fucosylation by lectins have been employed [13,22]. Major challenges with ELLA are the inherent glycosylation of the capture/detection antibody or non-specific binding by high-abundance glycoproteins from human matrices, which can cause a non-specific background signal by lectin detection, obscuring the analyte signal of interest.
Improved cancer specificity in PSA assay using Aleuria aurantia lectin coated Eu-nanoparticles for detection
2017, Clinical BiochemistryCitation Excerpt :AAL, as well as another fucose-binding lectin, i.e. Ulex europaeus I agglutinin (UEA-1; specific for fucα1-2Galβ), has been used to study the alterations in PSA fucosylation in PCa [11,21,23,24]. Methods used include either immuno-purification of PSA [11] or capturing and enrichment of fucosylated PSA as well as other glycoproteins using lectin affinity column chromatography [21,24]. Ohyama et al. reported that no significant differences in AAL bound fractions of PSA from PCa and BPH patient were found [21].