Prostate carcinoma is one of the most common causes of cancer-related deaths in men older than 50 years of age. It is predominantly a disease of older men, with a peak incidence between the ages of 65 and 75 years. Although the cause of prostate cancer is still unknown, clinical and experimental observations suggest that hormonal, genetic, diet and environmental factors, may play a role in its pathogenesis [1–6]. Carcinomas of the prostate are often clinically silent, especially during their initial stages, thus all efforts made towards early diagnosis and therapeutic investigations are important for the treatment of this malignant disease.

Cellular glycoconjugates are localised intracellularly, extracellularly and also at the cell surface, where they can be secretory or structural. Glycoproteins of the cell surface play different roles in cell function including cell differentiation, adhesion, proliferation, morphological changes and functional modulation. Glycosylation plays a major role by determining and stabilising protein folding, modelling of physicochemical properties and determining cell immunogenicity [7]. In the case of the prostate, complex glycans are important for the functional activity of this organ [8]. Glycoconjugates undergo modification associated with the cellular functions perform by them and also under pathological conditions. The glycosylation profile on tumour cells glycoproteins is distinctly different from that on normal cells, due to the blockage of the glycosylation pathways. This phenomenon results in the incomplete elongation of O-glycans and premature sialylation, leading to the expression of shorter and modified cancer-associated oligosaccharides such as T, sialyl-T, Tn, sialyl-Tn, LewisX (LeX) and sialyl-LeX, with a drastic impact in the cells normal function [9, 10].

Lectins are proteins of non-immune origin that reversibly bind specific carbohydrates and that may also agglutinate cells, precipitate polysaccharides or glycoconjugates [11]. They are widely used in biomedical diagnostic research as histochemical probes to localise and characterise specific carbohydrates residues or oligosaccharides in cells and tissues [12]. Lectin studies conducted in the human prostate, and in its secreted glycoproteins (e.g. PSA - prostate-specific antigen), have demonstrated their applicability as biomarkers of specific secretory functions, structural components and developmental alterations of this gland [13–16]. Additionally, lectins histochemical studies were performed to detect changes in glycoconjugates associated with pre-neoplasic and neoplasic transformation of the prostate [8, 17]. Frutalin is a tetrameric jacalin-related lectin expressed in the breadfruit plant seeds (Artocarpus incisa) that specifically recognises α-D-galactose residues [18]. Frutalin may be successfully used in immunobiological research, on the recognition of cancer-associated oligosaccharides, similarly to other galactose-binding lectins [19]. However, the use of lectins isolated from their natural sources presents several disadvantages, namely the heterogeneity of the sample due to the presence of lectin isoforms, which may lead to results variability. One way to overcome these limitations is a recombinant expression and production, which might improve and facilitate lectin availability and offer a higher level of purity and control of the lectin final properties.

In this work, the expression of frutalin specific glycoconjugates in human tissues of prostate carcinoma and benign prostate hyperplasia (BPH) was studied by immunohistochemistry. The binding pattern obtained with the breadfruit frutalin and with the recombinant frutalin produced by Pichia pastoris in the prostate tissues was compared and their potential use as tumour biomarkers was evaluated. For the first time a lectin from a recombinant source was used in immunohistochemical studies of human prostate tissues.

The major sugar residues present in the oligosaccharides of the cell surfaces glycoconjugates are: mannose, N-acetylglucosamine (GlcNac), N-acetylgalactosamine (GalNac), galactose (Gal), fucose (Fuc), and derivatives of sialic acid. The distinct property of lectins to bind specific carbohydrates makes them capable of recognising certain cell surface glycoproteins and detecting alterations on that, by using histochemical techniques. Various lectins have been used as biomarkers of human tumours, on the differentiation of dysplasic/neoplasic cells from normal/hyperplasic cells, as well as on the screening and prognosis of different types of carcinomas.

Several studies have shown that cell transformation into malignancy is closely related to an abnormal expression of carbohydrates on cell surfaces. A lectin immunohistochemical study suggested a strong expression of N-acetylgalactosamine residues in BPH and an increased expression of galactose, fucose and sialic acid residues in prostate carcinoma [8]. Thus, it would be expected that the galactose-binding lectin frutalin would recognize these modifications. Nevertheless, significant differences were found between the binding pattern of native and recombinant frutalin in the prostate tissues. Native frutalin bound to both hyperplasic and carcinoma tissues, while recombinant frutalin only bound to carcinoma tissues. However, the binding of recombinant frutalin to the neoplasic tissues was heterogeneous and did not occur in all studied cases. On the other hand, the binding of native frutalin was homogenous in all cases and very intense in the malignant ones. We have recently reported that native and recombinant frutalin presented a difference of at least 120-fold in carbohydrate-binding affinity [21]. The two lectins showed similar sugar-binding specificity but recombinant frutalin demonstrated less affinity. Thus, it can be concluded that it is the distinct carbohydrate-binding affinity presented by these two lectins that would lead to the different binding responses obtained. This is explained by the molecular differences found between native and recombinant frutalin [21]. Both are tetramer glycoproteins, but native frutalin is made of cleaved α and β chains, while in recombinant these two chains are connected by a linker tetrapeptide, because the cleavage of this linker did not occur in the P. pastoris expression system. It has already been reported that the linker cleavage may be needed to improve sugar-binding affinity of galactose-binding jacalin-related lectins [21, 23]. Moreover, the Pichia glycosylation pattern possibly altered the biological activity of recombinant frutalin as it inhibited the ability to agglutinate rabbit erythrocytes. In this study, recombinant frutalin has unequivocally distinguished between benign and malignant cells, binding exclusively to the neoplasic cells, in contrast to native frutalin. Thus, it can be concluded that the putative diagnostic value of recombinant frutalin may be higher than that for the native lectin. Native frutalin recognised all different histological prostate tissues possibly due to its higher carbohydrate-binding affinity and broad carbohydrate-binding range. In fact, native frutalin is a poly-specific lectin, as showed by sugar-binding fluorescence studies [21]. Native frutalin strongly reacted with the T-antigen disaccharide (Galβ1, 3GalNac) (binding constant in the 10⁴ M^-1 range) while no binding was observed with the recombinant frutalin. Furthermore, the affinity of recombinant frutalin to the monosaccharide Me-α-galactose is in the same magnitude order of the native frutalin affinity to other sugars that recombinant lectin did not recognise, as is the case of D-glucose (binding constants in the 10² M^-1 range). Therefore, the binding of frutalin to specific cancer-associated oligosaccharides could have been masked in prostate carcinoma tissues by the presence of other frutalin binding sugars. Nevertheless, the native frutalin binding responses suggest an increased expression of α-D-galactose residues on the neoplasic cells surface, as its binding was generally more intense in neoplasic cells than in hyperplasic cells, which corroborates previous studies [8].

Several lectins have been used in histochemical studies of the human prostate, to detect glycoconjugate changes related with the malignant transformation of this gland. The most studied lectins were the plant lectins soybean agglutinin (SBA - Glycine max agglutinin, specificity: α,βGal, α,βGalNAc) [8, 24–28], the gorse (furze) agglutinin (UEA-I - Ulex europaeus agglutinin, specificity: α-L-Fuc, GlcNAcβ1, 4GlcNAc) [8, 24, 26–30], and the peanut agglutinin (PNA - Arachis hypogaea agglutinin, specificity: Galβ1,3GalNAc) [8, 17, 24, 26–28, 31], which present different carbohydrate-binding specificities. Lectins from other organisms were also studied, such as the animal lectin HPA (garden snail - Helix pomatia agglutinin, specificity: αGalNAc, αGlcNAc, αGal) [8] and the fungal lectin AAL (mushroom - Aleuria aurantia lectin, specificity: α-L-Fuc) [8, 32]. Namely, several studies have demonstrated that the expression of HPA-binding glycoproteins on various human cancers, such as prostate carcinoma, is a marker of metastatic potential and poor prognosis [32].

The plant lectins SBA, UEA-I and PNA, as recombinant and native frutalin, were able to differentiate cells from distinct prostate histological tissues. Namely, SBA and UEA-I showed to be useful indicators of the prostate malignancy, as they distinguished between benign and malignant prostate cells, binding exclusively to dysplasic/carcinoma cells [24, 27, 28], or binding stronger to these cells, comparing to normal/hyperplasic cells binding [8, 26, 30]. However, in some studies SBA could not distinguish the referred cells [25, 26]. In addition, the PNA lectin bound stronger to neoplasic cells than to normal and hyperplasic cells [8, 17, 26, 27]. Nevertheless, negative binding in some malignant cases was also reported for these lectins, namely for the T-antigen specific PNA lectin (16% of total cases) [31].

It should be noted that all these lectins were obtained from their natural sources and therefore their availability is dependent from a time-consuming process and the final properties can vary as a result of different "batch to batch" isolations. This might have contributed to the variable and contradictory results above described. Frutalin, in particularly, is a heterogeneous mixture of several slightly different amino-acid sequences (isoforms), having or not different glycosylation places and extensions, while recombinant frutalin has a defined amino-acid sequence and therefore its properties are more effective. Frutalin isoforms may have different sugar-binding specificities/affinities and, consequently, variable results can be obtained. Furthermore, recombinant frutalin can be easily produced and purified in the P. pastoris heterologous expression system, which is an excellent way for frutalin large-scale production, considering its diagnostic application.

In this work, the binding of the frutalin lectin, obtained from its natural source and from a recombinant system, to prostate tissues was studied by immunohistochemistry. The results demonstrated that there are alterations in the carbohydrates of cellular glycoconjugates of benign and malignant human prostate tissues, detected by recombinant and native frutalin, namely enhancement of galactose residues in malignancy. The carbohydrate-binding properties of each lectin were responsible for the different binding patterns observed. Native frutalin bound stronger to neoplasic cells than to hyperplasic cells, while recombinant frutalin specifically recognised malignant cells. This study shows that both lectins have a considerable potential value in differentiating neoplasic changes from benign lesions of the human prostate. Namely, recombinant frutalin may be a useful tool to elucidate cases of difficult diagnosis. Moreover, the use of the P. pastoris recombinant system for frutalin production is more advantageous than the use of natural sources as the production and purification methodologies are easier and pure homogeneous frutalin samples are obtained.