A novel granulin homologue isolated from the jellyfish Cyanea capillata promotes proliferation and migration of human umbilical vein endothelial cells through the ERK1/2-signaling pathway.

Jellyfish grow rapidly and have a strong regenerative ability, indicating that they may express high levels of growth factors. Therefore, the aim of this research was to isolate the growth-promoting components from the jellyfish Cyanea capillata (C. capillata) and to further explore the underlying mechanisms. In this study, we first isolated and identified a novel polypeptide from C. capillata tentacles using size-exclusion chromatography followed by reverse-phase HPLC. This peptide, consisting of 58 amino acids (MW 5782.9 Da), belonged to the granulin (GRN) family of growth factors; thus, we named it Cyanea capillata granulin-1 (CcGRN-1). Second, using CCK-8 assay and flow cytometry, we verified that CcGRN-1 at the 0.5 µg/ml concentration could promote cell proliferation and increase the expression of cell-cycle proteins (CyclinB1 and CyclinD1). Third, signaling pathways studies showed that CcGRN-1 could activate the PI3K/Akt- and ERK1/2 MAPK-signaling pathways but not the JNK MAPK- or NF-κB-signaling pathways. Subsequently, we further confirmed that the CcGRN-1-induced cell proliferation and migration were associated only with the ERK1/2 MAPK-signaling pathway. Considering all of these factors, CcGRN-1, as the first jellyfish-derived GRN homologue, possesses growth-promoting properties and may be a candidate for novel therapeutics to promote human wound healing in unfavorable conditions.

Chromatographic Methods for the Purification of Granulin Peptides.

Progranulin is composed of seven repeating cysteine-rich granulin domains. In some cells and tissues, the progranulin is fragmented by proteolysis to generate the granulin modules as individual peptides, which are collectively referred to as granulins. These peptides are often biologically active, but the activity need not be identical to that of the parental progranulin from which they are derived. Thus, some granulin peptides stimulate cell proliferation, as does progranulin itself, while other granulin peptides suppress proliferation. Similarly, some granulin peptides promote inflammation even though progranulin itself suppresses inflammation. Investigating the structural and biological properties of granulin peptides is challenging. Here we discuss methods that employ reversed-phase high-performance liquid chromatography (RP-HPLC) and in some instances size-exclusion high-performance liquid chromatography (SE-HPLC) to isolate granulin peptides from tissues, in particular those that are rich in inflammatory cells such as neutrophils, bone marrow, or hematopoietic organs of teleost fish.

Large-Scale Generation of Recombinant Granulin Peptides in E. coli.

Generating milligram quantities of correctly folded granulin molecules with properly formed disulfide bonds and biologically relevant activities may represent a considerable challenge. Here I describe a protocol for obtaining well-folded human granulins A, C, and F by expressing them as thioredoxin fusion proteins in Origami (DE3) Escherichia coli cells promoting disulfide bond formation in the cytoplasm environment. The thioredoxin tag is removed by proteolytic cleavage with enterokinase and granulins which are purified by reversed-phase HPLC. Well-folded disulfide species display lower retention time than misfolded species and therefore can be readily purified.