Recombinant Bovine Membrane protein FAM159B (FAM159B)

What is FAM159B and what is its known functional role?

FAM159B is a transmembrane adaptor protein belonging to the Shisa-like protein family. The bovine FAM159B protein consists of 161 amino acids and has structural features consistent with transmembrane signaling functions. Although its exact function remains unknown, research suggests it acts as a transmembrane adaptor involved in regulating other transmembrane receptors and proteins . In pancreatic islets, FAM159B expression has been linked to β-cell exocytosis and maturation, suggesting a role in insulin secretion regulation . Vertebrates possess two copies of FAM159: FAM159A and FAM159B, with substantial differences in their amino acid sequences .

How do expression patterns of FAM159B differ between humans, rats, and mice in pancreatic islets?

The most prominent species difference in FAM159B expression is observed in pancreatic islets. Immunohistochemical studies reveal that while FAM159B expression is limited to single cells on the outer edges in mice and rats, it is detectable across entire islets in humans . Double-labeling experiments have shown that in human pancreatic islets, FAM159B expression partially overlaps with insulin-producing β-cells and, to a lesser extent, with glucagon-releasing α-cells . This supports a correlation between FAM159B and insulin expression .

In humans, FAM159B also shows partial overlap with somatostatin (SST) expression in Langerhans islets. Contrastingly, rat and mouse samples do not exhibit overlap between FAM159B and insulin or glucagon expression but show strong colocalization with SST expression . This suggests that in humans, FAM159B may be involved in the regulation of insulin secretion and modulation of glucagon and SST release, whereas in rodents, its role may differ significantly .

Interestingly, cellular localization also differs: in human samples, FAM159B expression was localized mainly at the cell membrane in insulin- and glucagon-containing cells, while in all three species, it was localized predominantly to the cytoplasm in SST-expressing cells . This suggests potential differences in functional state or even distinct functions across cell types.

How is FAM159B expression altered in diabetic conditions?

Research comparing lean (non-diabetic) and obese (diabetic) Zucker rats showed significant differences in FAM159B expression in pancreatic islets. The following table summarizes the key findings:

What molecular mechanisms might explain FAM159B's role in β-cell function?

While the exact mechanisms remain to be fully elucidated, research indicates that FAM159B is linked to β-cell exocytosis and maturation in pancreatic islets . The protein's transmembrane nature and its classification as an adaptor protein suggest it may function through protein-membrane or protein-protein interactions mediated by modular domains and/or peptide motifs .

FAM159B has been shown to coexpress with various neuroendocrine-specific markers and receptors, including chromogranin A, neuron-specific enolase, insulinoma-associated protein 1, neural cell adhesion molecule 1, dopamine receptor 2, regulator of G-protein signaling 9, and somatostatin receptors 2, 4, and 5 . This coexpression pattern suggests potential involvement in multiple signaling pathways relevant to neuroendocrine function.

The differential cellular localization of FAM159B (membrane-associated in insulin- and glucagon-containing cells versus cytoplasmic in somatostatin-expressing cells) further suggests that its subcellular distribution may be crucial for its specific functions in different cell types .

What are optimal methods for producing and storing recombinant bovine FAM159B protein?

Recombinant full-length bovine FAM159B protein can be successfully expressed in E. coli with an N-terminal His tag . The protein is typically provided as a lyophilized powder and should be reconstituted in deionized sterile water to a concentration of 0.1-1.0 mg/mL . For long-term storage, it is recommended to add 5-50% glycerol (with 50% being the default final concentration) and aliquot for storage at -20°C/-80°C .

Working aliquots can be stored at 4°C for up to one week, but repeated freezing and thawing should be avoided as this may compromise protein integrity . The reconstitution buffer typically consists of Tris/PBS-based buffer with 6% Trehalose at pH 8.0 . Before opening, vials should be briefly centrifuged to bring contents to the bottom .

What immunohistochemical approaches are effective for studying FAM159B expression?

Effective immunohistochemical protocols for studying FAM159B include:

• Tissue Preparation: Formalin-fixed, paraffin-embedded tissue sections (3-4 μm thick) .

• Antigen Retrieval: Microwave treatment in 10 mM citric acid (pH 6.0) for 16 minutes at 600W after blocking endogenous peroxidase activity with H₂O₂ in methanol .

• Primary Antibody: Anti-FAM159B antibody (such as HPA011778 from Atlas Antibodies) incubated overnight at 4°C .

• Detection System: Biotinylated secondary antibody followed by peroxidase-conjugated avidin (Vector ABC Elite Kit) with visualization using 3-amino-9-ethylcarbazole in acetate buffer .

• Controls: Specificity can be verified by preadsorbing the primary antibody with its immunizing peptide (e.g., PrEST Antigen FAM159B), which should result in complete loss of immunosignal .

For double-labeling experiments to study colocalization with other proteins (insulin, glucagon, or somatostatin), fluorescent-labeled secondary antibodies can be used:

• Cy3-conjugated anti-rabbit secondary antibody for FAM159B

• Alexa Fluor 488-conjugated anti-mouse or anti-rat secondary antibodies for other markers

• Mounting in Fluoromount G with DAPI for nuclear counterstaining

What considerations are important when designing comparative studies of FAM159B across species?

When designing comparative studies of FAM159B across species, researchers should consider:

• Antibody Cross-Reactivity: Ensure that antibodies can recognize the target protein across species. The polyclonal antibody HPA011778 recognizes human, rat, and mouse FAM159B due to sequence conservation at the C-terminal end .

• Species Differences: Account for known differences in expression patterns, particularly in pancreatic islets where FAM159B expression varies significantly between humans and rodents .

• Cellular Localization: Include subcellular localization analysis, as FAM159B shows different localization patterns (membrane vs. cytoplasmic) depending on cell type .

• Colocalization Studies: Design double-labeling experiments to assess coexpression with relevant markers like insulin, glucagon, and somatostatin due to species-specific differences in colocalization patterns .

• Controls: Include appropriate controls to verify antibody specificity, such as peptide adsorption tests .

• Functional Context: Consider physiological state (e.g., diabetic vs. non-diabetic) as this affects FAM159B expression patterns .

How should researchers interpret species differences in FAM159B expression for translational research?

The significant species differences in FAM159B expression patterns, particularly in pancreatic islets, present important challenges for translational research. Researchers should:

• Recognize that findings from rodent models may not directly translate to humans, especially regarding FAM159B's role in insulin secretion and glucose homeostasis .

• Note that in humans, FAM159B exhibits partial overlap with insulin- and glucagon-producing cells, whereas in rodents, it overlaps primarily with somatostatin-expressing cells .

• Consider that cellular localization differences (membrane in insulin/glucagon cells vs. cytoplasmic in somatostatin cells) suggest potential functional differences .

• When designing studies, include multiple species comparisons to establish which aspects of FAM159B function are conserved versus species-specific .

• Interpret data cautiously when extrapolating from animal models to human pathophysiology, particularly for diabetes and other pancreatic disorders .

These differences suggest that FAM159B may intervene in glucose homeostasis in humans in a more complex way than previously postulated, while in rodents, it may not be directly involved in insulin release regulation .

What are potential confounding factors in quantitative analysis of FAM159B expression?

When quantitatively analyzing FAM159B expression in tissue samples, researchers should consider:

What are promising approaches to elucidate FAM159B's functional role in pancreatic β-cells?

To better understand FAM159B's role in pancreatic β-cells, researchers might consider:

• CRISPR/Cas9 Studies: Generate FAM159B knockout or knock-in models to directly assess its role in insulin secretion and glucose homeostasis.

• Interactome Analysis: Identify protein binding partners of FAM159B in different cell types using co-immunoprecipitation followed by mass spectrometry.

• Live Cell Imaging: Track FAM159B dynamics during insulin secretion cycles to understand its temporal relationship with exocytosis.

• Conditional Expression Models: Develop inducible expression systems to study the acute effects of FAM159B modulation on cell function.

• Comparative Proteomics: Analyze differences in protein expression and phosphorylation states between wild-type and FAM159B-deficient cells to identify downstream signaling pathways.

How might FAM159B be involved in the pathophysiology of diabetes?

The altered expression of FAM159B in diabetic conditions suggests potential involvement in diabetes pathophysiology. Research shows that the area of FAM159B-positive cells as a percentage of total islet area is significantly larger in non-diabetic lean rats than in diabetic obese rats . This difference, combined with FAM159B's known association with β-cell exocytosis and maturation , suggests it may play a role in:

• β-Cell Function: Potential involvement in insulin secretion mechanisms that become dysregulated in diabetes.

• Islet Cell Adaptation: Possible role in adaptive responses of different islet cell populations during diabetes progression.

• Hormone Balance: Given its differential expression in insulin-, glucagon-, and somatostatin-producing cells across species, FAM159B might influence the balance between these hormones, which is disrupted in diabetes.

• β-Cell Survival: Potential contribution to mechanisms governing β-cell survival or apoptosis under metabolic stress conditions.

Further research investigating FAM159B expression in human diabetic samples and mechanistic studies in relevant model systems would help clarify its specific role in diabetes pathophysiology.