Voltage-gated sodium channels interact with cytosolic proteins that regulate channel trafficking and/or modulate the biophysical properties of the channels. Na(v)1.6 is heavily expressed at the nodes of Ranvier along adult CNS and PNS axons and along unmyelinated fibers in the PNS. In an initial yeast two-hybrid screen using the C terminus of Na(v)1.6 as a bait, we identified FHF2B, a member of the FGF homologous factor (FHF) subfamily, as an interacting partner of Na(v)1.6. Members of the FHF subfamily share approximately 70% sequence identity, and individual members demonstrate a cell- and tissue-specific expression pattern. FHF2 is abundantly expressed in the hippocampus and DRG neurons and colocalizes with Na(v)1.6 at mature nodes of Ranvier in myelinated sensory fibers in the dorsal root of the sciatic nerve. However, retinal ganglion cells and spinal ventral horn motor neurons show very low levels of FHF2 expression, and their axons exhibit no nodal FHF2 staining within the optic nerve and ventral root, respectively. Thus, FHF2 is selectively localized at nodes of dorsal root sensory but not ventral root motor axons. The coexpression of FHF2B and Na(v)1.6 in the DRG-derived cell line ND7/23 significantly increases the peak current amplitude and causes a 4 mV depolarizing shift of voltage-dependent inactivation of the channel. The preferential expression of FHF2B in sensory neurons may provide a basis for physiological differences in sodium currents that have been reported at the nodes of Ranvier in sensory versus motor axons.

FHF2A and FHF2B are two members of the fibroblast growth factor homologous factor 2 (FHF2) subfamily with distinct N termini. Using a generic antibody and electrophysiological methods, we previously showed that FHF2 is expressed in hippocampus and dorsal root ganglion (DRG) neurons and is colocalized with sodium channel Na(v)1.6 at sensory but not motor nodes of Ranvier, and that FHF2B associates with Na(v)1.6, causing an increase in current density and a small depolarizing shift in availability of channels. Using immunolabeling of adult rat tissue, we demonstrate that FHF2A is present within DRG but not in hippocampal or cerebellar neurons or at nodes of Ranvier in sciatic nerve, and that Na(v)1.6 and FHF2A are colocalized in nonmyelinated fibers. We also show that FHF2A binds directly to Na(v)1.6, and that the two proteins coimmunoprecipitate from transfected HEK293 cells. Because Na(v)1.6 has been associated with rapid firing rates, we examined the possible effects of FHF2B and the sister isoform, FHF2A, on electrophysiological properties of this channel in the DRG-derived ND7/23 cell line. We show that FHF2B inhibits accumulation of inactivation in response to trains of stimulation at high frequencies. In marked contrast, FHF2A causes an accumulation of inactivated channels at all frequencies tested due to a slowing of recovery from inactivation. Thus different FHF2 subfamily members have different functional effects on Na(v)1.6 and are differentially distributed in DRG neurons and their axons. This suggests that FHF2A and FHF2B may selectively alter firing behaviour of specific neuronal compartments via differential modulation of Na(v)1.6.

Fibroblast growth factor homologous factors (FHFs) form native intracellular complexes with the mitogen-activated protein kinase (MAPK) scaffold protein islet-brain 2 (IB2) in adult brain. FHF binding to IB2 facilitates recruitment of the MAPK p38delta (SAPK4), while failing to stimulate binding of JNK, the preferred kinase of the related scaffold IB1 (JIP-1). We now report further biochemical evidence supporting FHFs as regulators of IB2 scaffold activity. Mixed lineage kinase 3 (MLK3) and IB2 synergistically activate p38delta but not the MAPKs JNK-1 and p38alpha. Binding of p38delta to IB2 is mediated by the carboxyl-terminal half of the scaffold (IB2(Delta1-436)). FHF2 also binds weakly to IB2(Delta1-436) and can thereby increase p38delta interaction with IB2(Delta1-436). FHF-induced recruitment of p38delta to IB2 is accompanied by increased levels of activated p38delta, and synergistic activation of p38delta by MLK3 and IB2 is further enhanced by FHF2. Consistent with a role for FHFs as signaling molecules, FHF2 isolated from rat brain is serine/threonine-phosphorylated, and FHF can serve as a substrate for p38delta in vitro. These results support the existence of a signaling module in which IB2 scaffolds a MLK3/MKK/p38delta kinase cascade. FHFs aid in recruitment of p38 to IB2 and may serve as kinase substrates.

Börjeson-Forssman-Lehmann syndrome (BFLS) is a syndromal X-linked mental retardation, which maps by linkage to the q26 region of the human X chromosome. We have identified a male patient with BFLS-like features and a duplication, 46,Y,dup(X)(q26q28), inherited from his phenotypically normal mother. Fluorescence in situ hybridisation using yeast artificial chromosome clones from Xq26 localised the duplication breakpoint to an approximately 400-kb interval in the Xq26.3 region between DXS155 and DXS294/DXS730. Database searches and analysis of available genomic DNA sequence from the region revealed the presence of the fibroblast growth factor homologous factor gene, FHF2, within the duplication breakpoint interval. The gene structure of FHF2 was determined and two new exons were identified, including a new 5' end exon, 1B. FHF2 is a large gene extending over approximately 200 kb in Xq26.3 and is composed of at least seven exons. It shows tissue-specific alternative splicing and alternative transcription starts. Northern blot hybridisation showed highest expression in brain and skeletal muscle. The FHF2 gene localisation and tissue-specific expression pattern suggest it to be a candidate gene for familial cases of the BFLS syndrome and other syndromal and non-specific forms of X-linked mental retardation mapping to the region.

Cancer cells often develop drug resistance. In cisplatin-resistant HeLa cisR cells, fibroblast growth factor 13 (FGF13/FHF2) gene and protein expression was strongly upregulated, and intracellular platinum concentrations were kept low. When the FGF13 expression was suppressed, both the cells' resistance to platinum drugs and their ability to keep intracellular platinum low were abolished. Overexpression of FGF13 in parent cells led to greater resistance to cisplatin and reductions in the intracellular platinum concentration. These cisplatin-resistant cells also showed increased resistance to copper. In preoperative cervical cancer biopsy samples from poor prognoses patients after cisplatin chemoradiotherapy, FGF13-positive cells were detected more abundantly than in the biopsy samples from patients with good prognoses. These results suggest that FGF13 plays a pivotal role in mediating resistance to platinum drugs, possibly via a mechanism shared by platinum and copper. Our results point to FGF13 as a novel target and useful prognostic guide for cancer therapy.

CD133+ cells are hemangioblasts that have capacity to generate into both hematopoietic and endothelial cells (ECs). Hypoxia/normoxia has shown to be the regulator of the balance between stemness and differentiation. In this study we performed Agilent's whole human genome oligo microarray analysis and examined the differentiation potential of the bone-marrow-derived CD133+ cells after hypoxic/normoxic preconditioning of CD133+ cells. Results showed that there was no significant increase in erythroid colony forming unit (CFU-E) and CFU-granulocyte, erythrocyte, monocyte, and megakaryocyte formation with cells treated under hypoxia/normoxia. However, a significant increment of EC forming unit at 24 h (143.2 +/- 8.0%) compared to 0 h (100 +/- 11.4%) was observed in CFU-EC analysis. Reverse transcription-polymerase chain reaction and immunostaining analysis showed that the differentiated cells diminished hematopoietic stem cell surface markers and acquired the gene markers and functional phenotype of ECs. The transcriptome profile revealed a cluster of 232 downregulated and 498 upregulated genes in cells treated for 24 h under hypoxia. The upregulated genes include angiogenic genes, angiogenic growth factor genes, angiogenic cytokine and chemokine genes, as well as angiogenic-positive regulatory genes, including FGFBP1, PDGFB, CCL15, CXCL12, CXCL6, IL-6, PTN, EREG, ERBB2, EDG5, FGF3, FHF2, GDF15, JUN, L1CAM, NRG1, NGFR, and PDGFB. On the other hand, angiogenesis inhibitors and related genes, including IL12A, MLLT7, STAB1, and TIMP2, are downregulated. Taken together, hypoxic/normoxic preconditioning may lead to the differentiation of CD133+ cells toward endothelial lineage, which may improve the current clinical trial studies.

BACKGROUND

Fibroblast growth factors (FGFs) regulate the development of white adipose tissue (WAT). However, the secretion and cellular origin of individual FGFs in WAT as well as the influence of obesity are unknown.

OBJECTIVE

Our objective was to map FGFs in human sc WAT, the cellular source, and association with obesity.

METHODS

Secretion, mRNA, and circulatory levels of FGFs in human abdominal sc WAT from nonobese and obese donors were examined by microarray, real-time quantitative PCR, and ELISA. The activity of FGFs in cultured human adipocytes was determined by phosphorylation assays.

RESULTS

Expression of five FGFs (FGF1, FGF2, FGF7, FGF9, and FGF18) and FGF homologous factor (FHF2) was identified in WAT. Only FGF1 was released in a time-dependent manner from sc WAT, and fat cells were the major source of FGF1 secretion. FGF1 expression increased and FGF2 decreased during adipocyte differentiation. Furthermore, FGF1 was not secreted into the circulation. Although FGF1 levels were 2-fold increased in obesity, they were unaltered by weight reduction. Only FGF1 and FGF2 induced a marked concentration-dependent phosphorylation of p44/42 in cultured human adipocytes.

CONCLUSIONS

Of the investigated FGFs, only FGF1 is secreted from sc WAT and predominantly so from the adipocyte fraction. The activity in adipocyte cultures and lack of secretion into the circulation suggest that FGF1 acts as an auto- or paracrine factor. FGF1 levels are increased in obesity but unaffected by weight reduction, suggesting a primary defect in obese individuals. In conclusion, FGF1 may play a superior role among the FGFs in sc WAT and obesity development.

FGF13 (FHF2), the major fibroblast growth factor homologous factor (FHF) in rodent heart, directly binds to the C-terminus of the main cardiac sodium channel, NaV1.5. Knockdown of FGF13 in cardiomyocytes induces slowed ventricular conduction by altering NaV1.5 function. FGF13 has five splice variants, each of which possess the same core region and C terminus but differing in their respective N termini. Whether and how these alternatively spliced N termini impart isoform-specific regulation of NaV1.5, however, has not been reported. Here, we exploited a heterologous expression to explore the specific modulatory effects of FGF13 splice variants FGF13S, FGF13U and FGF13YV on NaV1.5 function. We found these three splice variants differentially modulated NaV1.5 current density. Although steady-state activation was unaltered by any of the FGF13 isoforms (compared to control cells expressing Nav1.5 but not expressing FGF13), open-state fast inactivation and closed-state fast inactivation were markedly slowed, steady-state availability was significantly shifted toward the depolarizing direction, and the window current was increased by each of FGF13 isoforms. Most strikingly, FGF13S hastened the rate of NaV1.5 entry into the slow inactivation state and induced a dramatic slowing of recovery from inactivation, which caused a large decrease in current after either low or high frequency stimulation. Overall, these data showed the diversity of the roles of the FGF13 N-termini in NaV1.5 channel modulation and suggested the importance of isoform-specific regulation.

Fever is a highly conserved systemic response to infection dating back over 600 million years. Although conferring a survival benefit, fever can negatively impact the function of excitable tissues, such as the heart, producing cardiac arrhythmias. Here we show that mice lacking fibroblast growth factor homologous factor 2 (FHF2) have normal cardiac rhythm at baseline, but increasing core body temperature by as little as 3 °C causes coved-type ST elevations and progressive conduction failure that is fully reversible upon return to normothermia. FHF2-deficient cardiomyocytes generate action potentials upon current injection at 25 °C but are unexcitable at 40 °C. The absence of FHF2 accelerates the rate of closed-state and open-state sodium channel inactivation, which synergizes with temperature-dependent enhancement of inactivation rate to severely suppress cardiac sodium currents at elevated temperatures. Our experimental and computational results identify an essential role for FHF2 in dictating myocardial excitability and conduction that safeguards against temperature-sensitive conduction failure.

Fibroblast growth factor homologous factors (FHFs) belong to the fibroblast growth factor (FGF) superfamily, which plays an important role in prostate cancer (PCa). Mining of public database suggests that FGF13 (FHF2) mRNA expression is altered in over 30% of PCa cases. This study examined the FGF13 expression pattern in human PCa specimens and evaluated its potential as a biomarker for patient outcome after radical prostatectomy (RP). Immunohistochemistry (IHC) showed that FGF13 was detectable in the majority of human PCa samples, and FGF13 IHC scores were higher in high-grade prostatic intraepithelial neoplasia, in primary PCa and in metastatic PCa than in benign prostatic tissue. There was a significant association between high cytoplasmic FGF13 staining and a risk of biochemical recurrence (BCR) after RP. This was also evident in the intermediate to high-risk patient groups. In contrast, positive nuclear FGF13 staining along with low cytoplasmic FGF13 group showed a decreased BCR risk. Multivariate regression analysis indicated that high cytoplasmic FGF13 staining was associated with BCR and that this could serve as an independent prognostic marker in PCa. Several PCa cell lines showed increased FGF13 expression at the mRNA and protein levels compared to the immortalized prostate epithelial cell line PNT1a. Analysis of co-labeled cells suggested a possible interaction of FGF13 with α-tubulin and the voltage-gated sodium channel proteins (Na(V)s/VGSCs). Our data indicate that, for PCa patients after RP, FGF13 serves as a potential novel prognostic marker that improves prediction of BCR-free survival, in particular if combined with other clinical parameters.

Nav1.6 and Nav1.6-mediated resurgent currents have been implicated in several pain pathologies. However, our knowledge of how fast resurgent currents are modulated in neurons is limited. Our study explored the potential regulation of Nav1.6-mediated resurgent currents by isoforms of fibroblast growth factor homologous factor 2 (FHF2) in an effort to address the gap in our knowledge. FHF2 isoforms colocalize with Nav1.6 in peripheral sensory neurons. Cell line studies suggest that these proteins differentially regulate inactivation. In particular, FHF2A mediates long-term inactivation, a mechanism proposed to compete with the open-channel blocker mechanism that mediates resurgent currents. On the other hand, FHF2B lacks the ability to mediate long-term inactivation and may delay inactivation favoring open-channel block. Based on these observations, we hypothesized that FHF2A limits resurgent currents, whereas FHF2B enhances resurgent currents. Overall, our results suggest that FHF2A negatively regulates fast resurgent current by enhancing long-term inactivation and delaying recovery. In contrast, FHF2B positively regulated resurgent current and did not alter long-term inactivation. Chimeric constructs of FHF2A and Navβ4 (likely the endogenous open channel blocker in sensory neurons) exhibited differential effects on resurgent currents, suggesting that specific regions within FHF2A and Navβ4 have important regulatory functions. Our data also indicate that FHFAs and FHF2B isoform expression are differentially regulated in a radicular pain model and that associated neuronal hyperexcitability is substantially attenuated by a FHFA peptide. As such, these findings suggest that FHF2A and FHF2B regulate resurgent current in sensory neurons and may contribute to hyperexcitability associated with some pain pathologies.

Fibroblast growth factor homologous factors (FHFs) are intracellular proteins which regulate voltage-gated sodium (Na_v) channels in the brain and other tissues. FHF dysfunction has been linked to neurological disorders including epilepsy. Here, we describe two sibling pairs and three unrelated males who presented in infancy with intractable focal seizures and severe developmental delay. Whole-exome sequencing identified hemi- and heterozygous variants in the N-terminal domain of the A isoform of FHF2 (FHF2A). The X-linked FHF2 gene (also known as FGF13) has alternative first exons which produce multiple protein isoforms that differ in their N-terminal sequence. The variants were located at highly conserved residues in the FHF2A inactivation particle that competes with the intrinsic fast inactivation mechanism of Na_v channels. Functional characterization of mutant FHF2A co-expressed with wild-type Na_vFHF2A proteins lost the ability to induce rapid-onset, long-term blockade of the channel while retaining pro-excitatory properties. These gain-of-function effects are likely to increase neuronal excitability consistent with the epileptic potential of FHF2 variants. Our findings demonstrate that FHF2 variants are a cause of infantile-onset developmental and epileptic encephalopathy and underline the critical role of the FHF2A isoform in regulating Na_v channel function.

Keywords: FGF13; FHF2; X linked; developmental and epileptic encephalopathy; epilepsy; epileptic encephalopathy; infantile onset; voltage-gated sodium channel.

Fibroblast Growth Factor Homologous Factors (FHF) constitute a subfamily of FGF proteins with four prototypes (FHF1-4; also known as FGF11-14). FHF proteins have been shown to bind directly to the membrane-proximal segment of the C-terminus in voltage-gated sodium channels (Nav), and regulate current density, availability, and frequency-dependent inhibition of sodium currents. Members of the FHF2 subfamily, FHF2A and FHF2B, differ in the length and sequence of their N-termini, and, importantly, differentially regulate Nav1.6 gating properties. Using immunohistochemistry, we show that FHF2 isoforms are expressed in adult dorsal root ganglion (DRG) neurons where they co-localize with Nav1.6 and Nav1.7. FHF2A and FHF2B show differential localization in neuronal compartments in DRG neurons, and levels of expression of FHF2 factors are down-regulated following sciatic nerve axotomy. Because Nav1.7 in nociceptors plays a critical role in pain, we reasoned that its interaction with FHF2 isoforms might regulate its current properties. Using whole-cell patch clamp in heterologous expression systems, we show that the expression of FHF2A in HEK293 cell line stably expressing Nav1.7 channels causes no change in activation, whereas FHF2B depolarizes activation. Both FHF2 isoforms depolarize fast-inactivation. Additionally, FHF2A causes an accumulation of inactivated channels at all frequencies tested due to a slowing of recovery from inactivation, whereas FHF2B has little effect on these properties of Nav1.7. Measurements of the Nav1.7 current in DRG neurons in which FHF2 levels are knocked down confirmed the effects of FHF2A on repriming, and FHF2B on activation, however FHF2A and B did not have an effect on fast inactivation. Our data demonstrates that FHF2 does indeed regulate the current properties of Nav1.7 and does so in an isoform and cell-specific manner.

Rationale: Fibroblast growth factor homologous factors (FHFs) are key regulators of sodium channel inactivation. Mutations in these critical proteins have been implicated in human diseases including Brugada syndrome, idiopathic ventricular arrhythmias, and epileptic encephalopathy. The underlying ionic mechanisms by which reduced sodium channel availability in Fhf2 knockout mice predisposes to abnormal excitability at the tissue level are not well defined. Objective: Using animal models and theoretical multicellular linear strands, we examined how FHF2 orchestrates the interdependency of sodium, calcium, and gap junctional conductances to safeguard cardiac conduction. Methods and Results: Fhf2^KO mice were challenged by reducing calcium conductance using verapamil or by reducing gap junctional conductance using carbenoxolone or by backcrossing into a connexin 43 heterozygous (Cx43^+/-) background. All conditions produced conduction block in Fhf2^KO mice, with Fhf2^WT showing normal impulse propagation. To explore the ionic mechanisms of block in Fhf2^KO hearts, multicellular linear strand models incorporating FHF2-deficient sodium channel inactivation properties were constructed and faithfully recapitulated conduction abnormalities seen in mutant hearts. The mechanisms of conduction block in mutant strands with reduced calcium conductance or gap junction uncoupling are very different. Enhanced sodium channel inactivation due to FHF2 deficiency shifts dependence onto calcium current to sustain electrotonic driving force, axial current flow, and action potential generation from cell-to-cell. In the setting of gap junction uncoupling, slower charging time from upstream cells conspires with accelerated sodium channel inactivation in mutant strands to prevent sufficient downstream cell charging for action potential propagation. Conclusions: FHF2-dependent effects on sodium channel inactivation ensure adequate sodium current reserve to safeguard against numerous threats to reliable cardiac impulse propagation.

Keywords: Fgf13; Fhf2; Shaw-Rudy model; fibro blast growth factor; impulse propagation.

Cancer therapy is often hampered by the disease's development of resistance to anticancer drugs. We previously showed that the autonomously upregulated product of fibroblast growth factor 13 gene (FGF13; also known as FGF homologous factor 2 (FHF2)) is responsible for the cisplatin resistance of HeLa cisR cells and that it is likely responsible for the poor prognosis of cervical cancer patients treated with cisplatin. Here we show that cloperastine and two other histamine H1 receptor antagonists selectively kill HeLa cisR cells at concentrations that little affect parental HeLa S cells. The sensitivity of HeLa cisR cells to cloperastine was abolished by knocking down FGF13 expression. Cisplatin-resistant A549 cisR cells were similarly susceptible to cloperastine. H2, H3, and H4 receptor antagonists showed less or no cytotoxicity toward HeLa cisR or A549 cisR cells. These results indicate that histamine H1 receptor antagonists selectively kill cisplatin-resistant human cancer cells and suggest that this effect is exerted through a molecular mechanism involving autocrine histamine activity and high-level expression of FGF13. We think this represents a potential opportunity to utilize H1 receptor antagonists in combination with anticancer agents to treat cancers in which emergent drug-resistance is preventing effective treatment.

The rodent heart is frequently used to study human cardiovascular disease (CVD). Although advanced cardiovascular ultrasound imaging methods are available for human clinical practice, application of these techniques to small animals remains limited due to the temporal and spatial-resolution demands. Here, an ultrasound vector-flow workflow is demonstrated that enables visualization and quantification of the complex hemodynamics within the mouse heart. Wild type (WT) and fibroblast growth factor homologous factor 2 (FHF2)-deficient mice (Fhf2KO/Y), which present with hyperthermia-induced ECG abnormalities highly reminiscent of Brugada syndrome, were used as a mouse model of human CVD. An 18-MHz linear array was used to acquire high-speed (30 kHz), plane-wave data of the left ventricle (LV) while increasing core body temperature up to 41.5°C. Hexplex (i.e., six output) processing of the raw data sets produced the output of vector-flow estimates (magnitude and phase); B-mode and color-Doppler images; Doppler spectrograms; and local time histories of vorticity and pericardium motion. Fhf2WT/Y mice had repeatable beat-to-beat cardiac function, including vortex formation during diastole, at all temperatures. In contrast, Fhf2KO/Y mice displayed dyssynchronous contractile motion that disrupted normal inflow vortex formation and impaired LV filling as temperature rose. The hexplex processing approach demonstrates the ability to visualize and quantify the interplay between hemodynamic and mechanical function in a mouse model of human CVD.

Developmental and epileptic encephalopathies (DEE) are a genetically heterogeneous group of disorders characterised by early onset epilepsy, epileptiform activity on electroencephalogram and associated developmental delay or neuroregression. With the advent of high throughput sequencing, novel gene-disease associations have been described for DEEs. Voltage activated sodium channels (Na_v) regulate neuronal excitability. Fibroblast growth factor homologous factors (FHFs) are proteins, which bind to the C terminal cytoplasmic tails of alpha subunits of Na_v channels and influence their function and surface expression. Gain of function hemizygous or heterozygous variants in FGF13 (also known as FHF2) were recently identified as the cause for X-linked developmental and epileptic encephalopathy 90 (DEE90; MIM# 301058) in seven individuals from five families, which included one female. We report an additional female, providing further evidence for a novel de novo heterozygous missense variant in FGF13, NM_004114.5: c.14T > G p.(Ile5Ser) causing X-linked DEE90. In addition, we review the genotype and phenotype of affected individuals with DEE90.

Keywords: EEG; FGFs; FHFs; Gain of function; Seizure; Sodium channels.

Fucoidanases are endo-fucoidanases (also known as endo-fucanases) that catalyze hydrolysis of α-glycosidic linkages in fucoidans, a family of sulfated fucose-rich polysaccharides primarily found in the cell walls of brown seaweeds. Fucoidanases are promising tools for producing bioactive fucoidan oligosaccharides for a range of biomedical applications. High sulfation degree has been linked to high bioactivity of fucoidans. In this study, a novel fucoidanase, Fhf2, was identified in the genome of the aerobic, Gram-negative marine bacterium Formosa haliotis. Fhf2 was found to share sequence similarity to known endo-α(1,4)-fucoidanases (EC 3.2.1.212) from glycoside hydrolase family 107. A C-terminal deletion mutant Fhf2∆484, devoid of 484 amino acids at the C-terminus, with a molecular weight of approximately 46 kDa, was constructed and found to be more stable than the full-length Fhf2 protein. Fhf2∆484 showed endo-fucoidanase activity on fucoidans from different seaweed species including Fucus evanescens, Fucus vesiculosus, Sargassum mcclurei, and Sargassum polycystum. The highest activity was observed on fucoidan from F. evanescens. The Fhf2∆484 enzyme was active at 20-45°C and at pH 6-9 and had optimal activity at 37°C and pH 8. Additionally, Fhf2∆484 was found to be calcium-dependent. NMR analysis showed that Fhf2∆484 catalyzed hydrolysis of α(1,4) linkages between L-fucosyl moieties sulfated on C2 (similar to Fhf1 from Formosa haliotis), but Fhf2∆484 in addition released oligosaccharides containing a substantial amount of 2,4-disulfated fucose residues. The data thus suggest that the Fhf2∆484 enzyme could be a valuable candidate for producing highly sulfated oligosaccharides applicable for fucoidan bioactivity investigations.

Keywords: FTIR; Fucus evanescens; Sargassum mcclurei; T9SS; calcium dependency; sulfation.