ST6GAL1 Human, sf9
Description
Structure and Production Overview
ST6GAL1 Human, sf9 is a glycosylated type II transmembrane protein belonging to glycosyltransferase family 29. Key structural features include:
The enzyme catalyzes the transfer of sialic acid (N-acetylneuraminic acid) from cytidine monophosphate (CMP) to terminal β-galactose residues on N-glycans, generating α2,6-sialic acid linkages .
Biochemical Properties and Activity
ST6GAL1 Human, sf9 exhibits enzyme kinetics suitable for in vitro studies:
The enzyme’s activity is quantified using a phosphatase-coupled assay, where released inorganic phosphate is detected via Malachite Green reagent . Proteolytic processing by BACE1 generates a soluble 364-amino-acid form, retaining catalytic activity within the Golgi lumen .
Research Applications
ST6GAL1 Human, sf9 is pivotal in studying α2,6-sialylation’s role in cancer, immunology, and cellular signaling:
Table 1: Key Research Applications
Glioblastoma (GBM)
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Role in Tumor-Initiating Cells (TICs): ST6GAL1 is enriched in GBM-derived BTICs, promoting α2,6-sialylation of PDGFRβ, which sustains phosphorylation and tumorigenic capacity .
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Therapeutic Targeting: Knockdown reduces BTIC self-renewal and in vivo tumor growth, suggesting ST6GAL1 inhibitors as a potential therapy .
B-Cell Precursor Acute Lymphoblastic Leukemia (BCP-ALL)
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Chemotherapy Resistance: Overexpression correlates with reduced vincristine sensitivity in ex vivo models. Knockdown paradoxically increases resistance, indicating context-dependent roles .
Mechanistic Insights
Product Specs
Q&A
What is ST6GAL1 and what is its primary function in human cells?
ST6GAL1 is the primary enzyme responsible for adding α2,6-linked sialic acid to N-glycosylated proteins destined for the plasma membrane or secretion . It catalyzes the transfer of sialic acid from CMP-sialic acid to galactose residues on glycoproteins, creating sialylated α2,6-lactosaminyl structures. This post-translational modification is critical for modulating cell signaling and behavior by affecting the structure and function of various cell surface receptors .
The enzyme is part of the ST6Gal family, which in higher vertebrates includes only two members: ST6Gal I and ST6Gal II . While ST6GAL1 (encoding ST6Gal I protein) is ubiquitously expressed across various tissues, ST6GAL2 shows a more restricted, tissue-specific expression pattern, primarily in adult brain and during embryonic development .
How is ST6GAL1 gene expression regulated in normal human tissues?
ST6GAL1 expression is regulated by multiple promoters governing the expression of several transcripts that encode identical polypeptide enzymes . This complex transcriptional regulation allows for tissue-specific expression patterns. High levels of ST6GAL1 mRNA are typically detected in hematopoietic cells and liver .
In normal tissues, ST6GAL1 protein expression appears to be particularly localized to stem/progenitor cell compartments. For example, in normal colon, ST6GAL1 localizes selectively to the base of crypts where stem/progenitor cells reside, with a staining pattern similar to the established stem cell marker ALDH1 . Similarly, in skin, ST6GAL1 expression is restricted to basal epidermal layers, another stem/progenitor cell compartment .
What are the validated methods for detecting ST6GAL1 protein expression?
Detection of ST6GAL1 has been challenging historically due to a lack of specific antibodies. Recent studies have validated effective antibodies that reliably detect ST6GAL1 protein . The following methods have been successfully employed:
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Immunoblotting/Western Blot: Using validated antibodies such as the goat anti-human ST6GAL1 antibody from R&D Systems (AF5924) . This antibody detects a specific band at approximately 56 kDa under reducing conditions .
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Immunohistochemistry: Paraffin-embedded tissue sections can be stained using validated antibodies following heat-induced epitope retrieval .
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Immunofluorescence: Cell cultures can be stained for ST6GAL1 using primary antibodies followed by fluorophore-conjugated secondary antibodies .
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Functional analysis: α2,6-sialylation can be assessed using the SNA (Sambucus nigra agglutinin) lectin, which specifically recognizes α2,6-linked sialic acids . This can be performed by flow cytometry using fluorescently-labeled SNA or by SNA precipitation assays followed by immunoblotting for specific target proteins .
How does ST6GAL1 contribute to cancer progression?
ST6GAL1 has been implicated in promoting tumorigenesis and cancer progression through several mechanisms:
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Increased expression in multiple cancers: ST6GAL1 is overexpressed in many cancer types including colon, breast, ovarian, and glioblastoma, with upregulation correlating with increased metastatic potential .
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Enhanced cell migration and invasion: In vitro studies demonstrate that ST6GAL1 promotes cell migration and invasion, partly through sialylation of the β1 integrin receptor .
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Regulation of cancer stem cells (CSCs): ST6GAL1 appears to be associated with the cancer stem cell phenotype. Selection of chemoresistant colon carcinoma cells leads to enrichment of CSCs (measured by CD133 and ALDH1 activity) with a corresponding upregulation of ST6GAL1 expression .
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Modulation of receptor signaling: ST6GAL1 regulates the levels and function of key receptors implicated in tumor growth, such as PDGF Receptor β (PDGFRB), Activated Leukocyte Cell Adhesion Molecule, and Neuropilin .
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Support of tumor growth: Knockdown of ST6GAL1 in brain tumor-initiating cells (BTICs) decreases in vitro growth, self-renewal capacity, and tumorigenic potential .
What role does ST6GAL1 play in stem cell biology?
ST6GAL1 shows a strong association with stem cell characteristics:
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Localization to stem cell compartments: In normal tissues like colon and skin, ST6GAL1 expression is confined to areas containing stem/progenitor cells .
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Expression in pluripotent stem cells: ST6GAL1 is highly expressed in induced pluripotent stem (iPS) cells, with no detectable expression in the fibroblasts from which the iPS cells were derived .
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Maintenance of stemness: Knockdown studies suggest that ST6GAL1 may be involved in maintaining stem-like cell behavior .
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Regulation of stem cell markers: Loss of ST6GAL1 can reduce expression of stem cell markers such as SOX2 .
What are the advantages of using sf9 cells for human ST6GAL1 expression?
While the search results don't specifically address sf9 expression systems for ST6GAL1, the advantages of this system for glycosyltransferases generally include:
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High expression yields: Sf9 insect cells infected with baculovirus can produce large amounts of recombinant protein.
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Post-translational modifications: Unlike bacterial systems, sf9 cells can perform many eukaryotic post-translational modifications, important for enzymes like ST6GAL1.
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Scalability: The sf9/baculovirus system can be readily scaled up for larger protein production needs.
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Lack of endogenous sialyltransferase activity: Insect cells naturally lack mammalian-type sialylation machinery, providing a "clean" background for functional studies of recombinant ST6GAL1.
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Protein folding: Sf9 cells provide an environment conducive to proper folding of complex mammalian proteins.
What are the common challenges in expressing functional human ST6GAL1 in sf9 cells?
Researchers should be aware of several challenges:
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Glycosylation differences: Insect cells produce different glycosylation patterns than mammalian cells, which may affect ST6GAL1 stability or activity.
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Need for CMP-sialic acid: For functional assays of expressed ST6GAL1, the donor substrate CMP-sialic acid must be provided, as insect cells typically don't produce significant amounts.
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Protein localization: ST6GAL1 is normally a Golgi-resident enzyme, so proper targeting within the sf9 cellular compartments needs to be considered in construct design.
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Proteolytic processing: Careful monitoring for potential proteolytic cleavage is necessary, as ST6GAL1 can exist in both full-length and cleaved forms .
How can we assess the functional activity of recombinant ST6GAL1?
Multiple approaches can be employed to determine if recombinant ST6GAL1 is enzymatically active:
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SNA lectin binding assays: Using SNA-FITC in flow cytometry to detect α2,6-sialylation of cell surface glycoproteins .
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Immunoprecipitation with SNA-agarose: Precipitation of α2,6-sialylated proteins followed by immunoblotting for specific target proteins of interest .
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In vitro sialylation assays: Using purified enzyme with CMP-sialic acid donor and appropriate acceptor substrates, followed by methods to detect sialic acid transfer.
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Target protein functional changes: Assessing changes in target receptor function, such as PDGFRB phosphorylation in response to PDGF-BB stimulation .
What experimental approaches are effective for studying ST6GAL1-mediated sialylation of specific target proteins?
The following methodologies have proven effective:
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SNA lectin precipitation: Tissue or cell homogenates can be incubated with SNA-agarose to isolate α2,6-sialylated proteins, which are then analyzed by immunoblotting for specific proteins of interest .
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ST6GAL1 knockdown/overexpression: Genetic manipulation of ST6GAL1 expression followed by functional assessment of target proteins, as demonstrated in studies with PDGFRB .
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Mass spectrometry: To identify specific glycosylation sites and confirm the presence of α2,6-linked sialic acids on target glycoproteins.
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Cell-based assays: Following manipulation of ST6GAL1 expression, functional assays can detect changes in cellular properties such as growth, migration, or response to ligands .
How does the evolutionary history of ST6GAL1 inform its functional analysis?
Phylogenomic analyses reveal important insights about ST6GAL1 evolution:
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Accelerated evolution: Evidence suggests accelerated evolution of st6gal1 genes in their genomic regulatory sequences and coding sequences in reptiles, birds, and mammals (amniotes) .
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Evolutionary divergence timing: The divergence between ST6GAL1 and ST6GAL2 in vertebrates has been established through molecular clock analyses using regression equations between linearized branch lengths and calibrated divergence dates .
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Expression pattern differentiation: ST6GAL1 shows a more ubiquitous expression pattern in higher vertebrates, while ST6GAL2 maintains a more ancestral, restricted expression profile throughout vertebrate evolution .
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Functional specialization: The evolutionary history suggests that ST6GAL1 has undergone neofunctionalization in higher vertebrates, potentially explaining its diverse roles in various physiological and pathological processes .
What strategies can be employed to target ST6GAL1 function in experimental or therapeutic settings?
Several approaches could be considered:
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RNA interference: shRNA-mediated attenuation of ST6GAL1 has been successfully used to decrease ST6GAL1 expression in cancer cells, leading to reduced numbers of CD133/ALDH1-positive cancer stem cells .
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CRISPR/Cas9 gene editing: For complete knockout or mutation of specific domains to study structure-function relationships.
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Small molecule inhibitors: Development of specific inhibitors targeting ST6GAL1 enzymatic activity.
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Competitive substrate analogs: Using modified versions of CMP-sialic acid that compete with the natural substrate.
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Targeting downstream effectors: Focusing on key receptors that are modified by ST6GAL1, such as PDGFRB in glioblastoma .
Product Science Overview
ST6 Beta-Galactosamide Alpha-2,6-Sialyltransferase 1 (ST6GAL1) is an enzyme that belongs to the glycosyltransferase family 29. This enzyme is responsible for the transfer of sialic acid from CMP-sialic acid to galactose-containing substrates, forming NeuAcα2,6-Gal linkages in N-linked glycans . The recombinant form of this enzyme, expressed in sf9 insect cells, is used for various research and therapeutic purposes.
ST6GAL1 was originally purified from rat liver tissues to homogeneity, which led to the identification of its cDNA . The enzyme was first described as ST6N and later termed Siat1 by the International System for Gene Nomenclature . The human recombinant form of ST6GAL1 is expressed in sf9 insect cells, which are derived from the fall armyworm Spodoptera frugiperda. This expression system is widely used for producing recombinant proteins due to its high yield and proper post-translational modifications.
ST6GAL1 is a type II membrane protein with a calculated molecular mass of approximately 43.5 kDa . The enzyme catalyzes the transfer of sialic acid to galactose-containing substrates, which is a crucial step in the biosynthesis of glycoproteins and glycolipids . This sialylation process is essential for various biological functions, including cell-cell interactions, immune responses, and pathogen recognition.
The enzyme shows a broad tissue distribution, with particularly high expression in the liver, lactating mammary gland, hematopoietic activated B cells, and subsets of T cells . Deficiency in ST6GAL1 results in a lack of α2,6-sialylation in N-linked glycans, which can lead to various biological consequences . The enzyme’s activity is also implicated in the generation of cell-surface carbohydrate determinants and differentiation antigens such as HB-6, CD75, and CD76 .
Recombinant ST6GAL1 expressed in sf9 cells is used in various research applications, including the study of glycosylation processes, the development of therapeutic glycoproteins, and the investigation of disease mechanisms related to sialylation . The enzyme’s role in immune responses and pathogen recognition makes it a valuable tool for studying infectious diseases and developing vaccines.
