Identification, Characterization, and Functional Study of the Two Novel Human Members of the Semaphorin Gene Family

Xianghu Qu,Handong Wei,Yun Zhai,Haiping Que,Qian Chen,Fei Tang,Yan Wu,Guichun Xing,Yunping Zhu,Shaojun Liu,Ming Fan,Fuchu He

doi:10.1074/jbc.m206451200

Xianghu Qu, Handong Wei + Show 10 more

Open Access

https://doi.org/10.1074/jbc.m206451200

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Abstract

We cloned two novel human transmembrane semaphorins, (HSA)SEMA6C and (HSA)SEMA6D, that belong to the class VI subgroup of the semaphorin family. The genes for SEMA6C and SEMA6D are mapped on chromosome 1q12-21.1 and 15q21.1, respectively. Among the adult tissues, SEMA6C is expressed only in skeletal muscle, whereas SEMA6D is expressed abundantly in kidney, brain, and placenta and moderately in the heart and skeletal muscles. During murine development, neither SEMA6C nor SEMA6D was expressed in embryonic day 10.5 (E10.5) embryos, but both were highly expressed in the areas of the lateral ventricle, the striatum, the wall of the midbrain, the pons/midbrain junction, and the choroid plexus of E13 embryos. Were neurons, neither axons nor astrocytes, highly expressed both semaphorins. Three isoforms of SEMA6C and five isoforms of SEMA6D derived from alternative splicing were identified, and their expression was regulated in a tissue- and development-dependent manner. Deletion analysis indicated that a sema domain and a PSI domain are integrally necessary for correct post-translation modification and subcellular localization. The extracellular domain of SEMA6C inhibited axonal extension of nerve growth factor-differentiated PC12 cells and induced the growth cone collapse of chicken dorsal root ganglion, rat hippocampal neurons, and rat cortical neurons in a dose-responsive manner. SEMA6D acted like SEMA6C except it had no significant effect on the growth cones of rat cortical neurons.

Highlights

We cloned two novel human transmembrane semaphorins, (HSA)SEMA6C and (HSA)SEMA6D, that belong to the class VI subgroup of the semaphorin family
Three isoforms of SEMA6C and five isoforms of SEMA6D derived from alternative splicing were identified, and their expression was regulated in a tissue- and development-dependent manner
The results presented above were confirmed by an additional dot blot analysis, in which a dot blot containing a total of 68 normal tissues and 8 human cancer lines was hybridized to the same probes for SEMA6C or SEMA6D, respectively

Summary

EXPERIMENTAL PROCEDURES

Identification and cDNA Cloning of Human SEMA6C and SEMA6D—Based on a cDNA clone (No FLD6219) homologous to rat Sema6C cDNA, we obtained a putative full-length cDNA of SEMA6C using contig from public data bases, PCR with primers directly based on the rat Sema6C sequence, and the 5Ј-RACE (rapid amplification of cDNA ends) technique (SMARTTM RACE cDNA Amplification Kit, CLONTECH). Two sets of primers were used to confirm existence of the predicted cDNA sequence of SEMA6C and to amplify its coding sequence (CDS) from human brain cDNA These resulted in pGEM-T vectors with the entire CDSs of the three alternative splicing variants (SEMA6C.1, SEMA6C.2, and SEMA6C.3), respectively. After a 60-min blocking at room temperature in 5% normal goat serum in buffer 2 (1% blocking reagent in buffer 1; Roche Molecular Biochemicals), embryos were incubated overnight at 4 °C in a 1:2000 dilution of anti-digoxigenin Fab fragment in 5% normal goat serum in buffer 2 They were washed four times with buffer 1 for 1 h each, washed twice with alkaline phosphatase buffer (0.1 mmol/liter Tris-HCl, pH 9.5, 0.1 mmol/liter NaCl, and 50 mmol/liter MgCl2) for 1 h each.

Two Novel Members of Class VI Semaphorin

RESULTS

DISCUSSION