VCAM1 Human, sf9

Basic Research Questions

• What is VCAM1 and what is its role in cellular adhesion?

  VCAM1 (Vascular Cell Adhesion Molecule 1) is a 110-kD member of the immunoglobulin gene superfamily expressed on the surface of cytokine-stimulated endothelial cells. It functions as an inducible adhesion receptor for circulating mononuclear leukocytes and certain tumor cells, mediating cell-to-cell adhesion events implicated in inflammation, cancer metastasis, and atherosclerosis development . VCAM1 contains seven C2-type immunoglobulin-like domains in its extracellular portion, each stabilized by disulfide bonds, followed by a transmembrane domain and cytoplasmic tail . The molecule specifically binds to the integrin VLA4 (α4β1) on leukocytes, facilitating important physiological and pathological adhesion processes . Unlike some adhesion interactions, VCAM1-VLA4 binding does not require divalent cations like Ca²⁺ or Mg²⁺, making it distinct from other adhesion mechanisms .

• Why is the Sf9 insect cell system commonly used for recombinant VCAM1 expression?

  The Sf9 insect cell system, part of the Baculovirus Expression Vector System (BEVS), offers several methodological advantages for VCAM1 expression:

  The BEVS produces properly folded proteins with correct post-translational modifications, essential for VCAM1's complex structure . This system accommodates large gene insertions like the full-length VCAM1 and offers high safety, operational simplicity, and adaptability for serum-free culture . Importantly, the expressed recombinant VCAM1 (rVCAM1) can be partially purified from membrane preparations while maintaining native conformation, preserving biological activity for functional assays .

  Despite these advantages, researchers should recognize the system's limitations, including potential expression instability and reduced protein glycosylation complexity compared to mammalian systems . The BEVS has demonstrated significant value in biotechnology, with eleven approved products including vaccines and therapeutics, making it a well-established platform for research applications requiring functional VCAM1 .

• What are the structural characteristics of human VCAM1?

  Human VCAM1 exhibits several distinct structural features essential for its function:

  It is a 100-110 kDa, 715 amino acid residue, type I transmembrane glycoprotein with an extracellular N-terminus . The extracellular portion contains seven C2-type immunoglobulin-like domains and extends approximately 674 amino acid residues . The molecule continues with a 22 amino acid transmembrane domain and concludes with a short 19 amino acid cytoplasmic tail .

  Each C2 domain contains a pair of cysteines forming stabilizing disulfide linkages, with additional disulfide bonds between extra cysteines associated with domains 1 and 4 . The protein contains multiple N-linked glycosylation sites critical for proper folding and function . Electron microscopy studies of related adhesion molecules suggest VCAM1 has a rod-like extracellular structure approximately 190 Å long with a potential kink between domains . This structural arrangement facilitates appropriate presentation of binding domains for interaction with counter-receptors.

• How do VCAM1-mediated adhesion mechanisms differ between cell types?

  VCAM1-mediated adhesion exhibits cell type-specific differences in mechanism and structure:

  When eosinophils adhere to VCAM-1, they form punctate adhesions called podosomes, which are highly dynamic structures containing β1 integrin subunit, phosphotyrosine-containing proteins, punctate filamentous actin, and gelsolin (a podosome marker) . In contrast, non-transformed fibroblasts on VCAM-1 develop peripheral focal adhesions positive for α4, β1, phosphotyrosine, vinculin, talin, and paxillin, but notably negative for gelsolin and associated with microfilaments rather than punctate actin .

  These cell-specific differences have functional implications. Eosinophil podosomes are associated with VCAM-1 degradation through metalloproteinase activity, specifically a disintegrin and metalloproteinase domain 8 (ADAM8) . This degradation can be inhibited by metalloproteinase inhibitors like ortho-phenanthroline . These distinct adhesive mechanisms may regulate cell-specific processes like eosinophil arrest and extravasation in allergic conditions, highlighting the importance of considering cell type when studying VCAM1-mediated adhesion.

• What are the basic steps for expressing functional human VCAM1 in Sf9 cells?

  Expressing functional human VCAM1 in Sf9 cells involves a systematic process:

  First, clone the full-length human VCAM1 cDNA into a baculovirus transfer vector under a strong promoter, typically polyhedrin (pH) or p10 . These promoters can be enhanced by elements like homologous region 1 (hr1), which increases protein expression 3-4 times in both Sf21 and Tni insect cells .

  Next, generate recombinant baculovirus by co-transfecting Sf9 cells with the transfer vector and linearized baculovirus DNA, allowing homologous recombination . After isolating and amplifying the recombinant virus, infect fresh Sf9 cells at an appropriate multiplicity of infection (MOI) .

  Harvest cells 48-72 hours post-infection when expression peaks, then prepare membrane fractions containing the expressed VCAM1 . This preparation can be used directly in adhesion assays or further purified while maintaining the protein's native conformation . Verify expression through Western blotting with anti-VCAM1 antibodies and test functionality using adhesion assays with cells expressing VLA4, such as Jurkat T cells .