Cell surface sialic acids convey many cellular processes ranging from cell-cell recognition to stabilization of glycoproteins. Abnormal sialylation patterns have been show in many cancer and malignancies. This altered cell surface sialylation is thought to arise from changes in the Golgi resident sialyltransferases but recent experiments have shown that altered flux in the sialic acid producing pathway can also influence the cell surface representation of sialic acids. In the present study we aimed to characterize the proteomic changes that occur after 24 hours of sialic acid, Neu5Ac, overproduction and to elucidate the altered functional processes that are affected by the increase in Neu5Ac production.
Based on previous study  we used HEK293 cell line to examine the effect of excess Neu5Ac on the cellular proteome. The production of Neu5Ac was induced by adding 30 mM of N-Acetylmannose to cell culture media. We used high mannose concentration and 24 hours of induction in order to elicit a large production of Neu5Ac and subsequent strong proteomic response in HEK293 cells without significant adverse effects on cell proliferation . Additionally, the long time period of induction should allow an adequate time for proteomic changes to occur and cells to adapt to the increased Neu5Ac concentration.
The ManNAc addition lead to almost immediate increase in cellular ManNAc levels as the concentration of ManNAc began to rise within the first fifteen minutes after induction (Figure 2). However after one hour the ManNAc levels seemed to stabilize to about two to three times higher than in the zero hour sample. This small increase of ManNAc is quite low compared to the excess of added extracellular ManNAc suggesting a regulation of intracellular ManNAc either by reduction of intake from environment or by consumption by ManNAc utilizing enzymes. In contrast to the cellular ManNAc levels, Neu5Ac concentration did not change within the first 30 minutes but began to steadily rise after one hour. The Neu5Ac levels continued to grow linearly for the next 24 hours to almost 70-fold while ManNAc levels increase only 4.8-fold compared to the zero hour samples.
The relative protein quantification analysis identified 105 significantly up- or down-regulated proteins in ManNAc induced cells. Quite surprisingly only seven of the significantly changed proteins showed up-regulation while 100 indicated reduction in abundance. BiNGO analysis of the 105 significantly changed proteins indicated reduction in the abundance Regulation of biological process, Transport, Plasma Membrane and Signal transduction annotated proteins. This suggests that the global adaptation to high ManNAc and Neu5Ac levels is achieved by reducing selected members of cellular transport machinery along with associated plasma membrane and signaling proteins. Such down-regulation could be used to modify the transport to or from plasma membrane in order to reduce the transport of overly sialylated proteins to cell surface or to inhibit the ManNAc transport within the cell.
Further functional analysis of the proteins revealed that the vesicular protein transport was affected by the ManNAc induction and Neu5Ac overproduction (Additional file 3). We identified 18 proteins that were annotated to protein transport categories, most of which were down-regulated. This finding may indicate that the response to high ManNAc and Neu5Ac concentration and possibly to subsequent alterations to cell surface sialylation is regulated by altering the cellular protein transport machinery. Interestingly we identified also eight small GTPases (RAB5A, RAB5C, RAB6B, RAB13, SAR1A, SAR1B, ARF5, and ARF4) in the protein transport- related proteins, seven of which were down- and one up-regulated. Small GTPases are a family of proteins that regulate the targeting and transport on endocytic protein transport vesicles. Small GTPases and their regulating proteins have been shown to confer specify of the cellular transport and also organelle identity [27–29]. Six of identified small GTPases are also annotated to Golgi apparatus. Golgi functions as the main factory of post-translationally modified cell surface proteins and requires several regulating molecules, including small GTPases, to maintain the dynamic transportation routes to and from the apparatus . The protein glycosylation machinery resides in Golgi, so increased sialylation flux within the Golgi and the following regulation of the Golgi transport machinery is not unexpected. It remains unknown whether the reduction of many small GTPases and other transport proteins means reduction on overall transport of cell surface proteins or alterations to specific transport routes within the cellular compartments.
Another major finding in the functional analyses was the reduction in several proteins (RAB5A, RAB5C, DNM2, TUBB1, ACTN2, NME1, TUBB4A, MAPRE1, ACTN3) associated with epithelial adheres junctions. Adherence junctions are plasma membrane bound protein complexes that mediate cellular contacts between cells through sialic acid containing transmembrane glycoproteins, E-cadherins [31–33]. The tightness and stability of adherens junctions between neighboring cells has been shown to be dependent on the cellular density of growing epithelial cell populations . The modifications in junction stability are thought to arise from alterations in E-cadherin glycosylation pattern . The observed changes in the Remodeling of adheres junction pathway proteins after ManNAc induction and Neu5Ac overproduction may be the result of altered sialylation of E-cadherin or other adherens junction proteins. Additionally, the recycling E-cadherin by endosomal transportation route may be altered as we identified E-cadherin recycling related proteins RAB5, DYN2 and NME1 in the set of down-regulated proteins [35, 36]. Several signaling pathways that are connected to remodeling of adhesion junctions pathway were also enriched supporting the findings that cell-cell contact points communicate with cellular processes through different signaling routes .
The impact of high ManNAc and Neu5Ac overproduction was seen on cellular proliferation level as several interconnected cell cycle, apoptosis and protein translation related signaling pathways were identified in the set of changed proteins (Figure 3). Additionally, ManNAc induction and overproduction of Neu5A affects the metabolic networks for nucleotide production. Purine and pyrimidine biosynthesis pathway proteins, including two committed step catalyzing enzymes of purine biosynthesis pathway (PPAT  and ADSS ), were reduced after induction with ManNAc. Even though the observed small reduction on cell proliferation was not statistically significant it is possible that the increase in ManNAc and Neu5Ac concentration causes additional strain on the cell population leading to reduced proliferation and modifications to cell growth related signaling processes. Reduced growth in turn leads to reduction in the requirement of nucleotides which is then controlled by proteomic down-regulation of control point proteins of these pathways.
Examination of physical interactions revealed several interacting protein clusters between changed proteins (Figure 4). Among these clusters were several two-protein complexes between protein paralogs, but also three multicomponent clusters with functional similarities between interaction partners. For example, we showed similar down-regulation of interacting proteins MAT2A and MAT2B . S-adenosylmethionine production by MAT2A has been shown to be regulated by association with MAT2B . The association of MAT2A and MAT2B also shields the MAT2A enzyme from proteasomic degradation thus affecting the cellular SAM levels . Additionally, we identified spliceosome- associated (NHP2L1, LSM2, LSM3, WDR77, ILF2), proteasomic (PSMC6 and USP14) and ribosomal (RPL7A, RPL11 and RPS3A) proteins that show changes in abundance. Such alterations to protein members of spliceosome, ribosome and proteasome may indicate regulation of the function or assembly of the respective multiprotein complexes.