We report here an initial characterization of the Rab27b gene in mammals. The human RAB27B
gene is found to consist of 6 exons spread over about 69 kb. The first, non-coding, exon is separated from the others by 49 kb and the end of translation is located within exon 6. This structure is very similar to the human RAB27A
], the exon/intron junctions are present at the equivalent positions and there is high sequence similarity (67% identity at the DNA level and 72% in the protein). It is likely that a duplication event has occurred to generate the two genes encoding the two isoforms of Rab27. Unlike RAB27A
however, exon 6 in RAB27B
has a very long (6.4 kb) 3' untranslated region but the significance of this finding is unclear at present.
The sequence of the human RAB27B
cDNAs and genomic DNA that we have analysed is found to differ slightly from that previously described by Chen et al. [8
]. One important difference affects the putative translation of the gene as it occurs in the coding region. We found that codon 206 is TTG (leucine) rather than the reported TGG (tryptophan). We sequenced several independent cDNA clones and genomic clones and our sequence matches five ESTs (Genbank AI432357, AW664866, AA812916, AW370404 and BF088963) in the database that overlap in the region where there is disparity with the Chen et al. [8
The mouse rab27b cDNA shows 89% at the DNA and 95% at the protein level with the human RAB27B cDNA. On Northern analysis, the mouse mRNA is only 2.5 kb in size which is considerably smaller than the 8 kb human mRNA. This smaller mRNA size corresponds with the 3' ends determined by a large number of mouse ESTs and is likely due to a much shorter 3' UTR in the mouse compared to the human gene.
Our expression analysis by RT-PCR suggests that Rab27b is expressed at lower levels than Rab27a. However, the PCR was not done under quantitative conditions and this could be the result of different rates of amplification of the different PCR fragments. On the other hand, both mRNAs were detected at approximately the same levels on Northern blots. Our Northern blots of mouse tissues indicated that mouse Rab27a is expressed in heart, spleen, lung and kidney while Rab27b is expressed in heart, brain, spleen and kidney. This distribution is somewhat different from our RT-PCR results for mouse tissues but this could be due to the presence of contaminating blood in the tissues used for analysis as Rab27 proteins are expressed in haemopoietic cell types. It remains to be established whether Rab27b is expressed in melanocytes. The initial cloning of Rab27b by RT-PCR from human melanoma cells [22
] would suggest that it is but our RT-PCR analysis on several melanocyte cell lines proved negative. Future studies with specific antibodies should clarify this issue.
The sequences of the human and mouse Rab27a and Rab27b genes confirm the extensive similarity between the two proteins. This suggests that these two Rab27 isoforms may be functional homologues which are active in different tissues. One would then expect the two proteins to be targeted to the same intracellular location in the same cells. We decided to study the subcellular localization of Rab27a and Rab27b in melanocytes as we have recently shown a very specific association of Rab27a with melanosomes, the specialised pigment-containing structures in these cells [16
]. Upon expression of GFP-Rab27 fusion proteins in melanocytes, we found that GFP-Rab27b, like GFP-Rab27a, co-localises to melanosomes. This suggests that, although Rab27b does not seem to be expressed in melan-c cells, it contains targeting determinants as present in Rab27a. However, further experiments are required to test whether there is functional redundancy between the two proteins.
Mutations in Rab27a in humans (Griscelli Disease) and mice (ashen
) result in melanosome transport and secretion defects [10
]. The lack of phenotype in the other tissues where Rab27a is expressed could be due to functional redundancy between the Rab27a and Rab27b proteins such that there is enough Rab27b in most tissues to complement the lack of Rab27a except in melanocytes where Rab27b was not detectable by RT-PCR.
We had previously implicated Rab27a in the pathogenesis of Choroideremia (CHM), an X-linked slow-onset retinal degenerative disease resulting from loss-of-function mutations in Rab Escort Protein-1 [7
]. As the CHM phenotype is very different from Griscelli Disease, it remains unclear whether Rab27a has any role on eye disease. It is unclear at present whether Griscelli patients develop retinal disease as the disease has a poor prognosis and patients don't survive past childhood [12
]. Also, the equivalent mutation in mice, ash
arose in the C3H strain homozygous for the rd
mutation and all C3H mice are blind a few weeks after birth [23
]. A likely possibility is that both Rab27 isoforms are partially affected in CHM and therefore contribute, possibly with other Rabs to the initiation and propagation of the degenerative process in CHM.
We show that the human RAB27B
gene maps to chromosome 18q21.1 by radiation hybrid mapping and FISH. This region of human 18q21.1 is syntenic with two mouse chromosomal regions in chromosomes 1 and 18. As the leaden
) mouse mutant locus maps to mouse chromosome 1 next to Bcl2
(which maps to 18q21.3 in humans), we investigated whether Rab27b
could be involved in ln
. The ln
mutation attracted our attention because ln
is phenotypically very similar to ash,
which is caused by mutations in Rab27a
which is caused by MyoVa
]. We therefore hypothesized that Rab27b was a candidate gene for leaden
. However, the mapping of mouse Rab27b to chromosome 18 suggests that Rab27b is not responsible for leaden
maps to chromosome 1. If Rab27a and Rab27b are indeed functional homologues, we would expect Rab27b mutations to cause deficiency in lysosomal transport in tissue(s) other than melanocytes where Rab27b, but not Rab27a, is expressed. The studies presented here will aid the future analysis of a possible association of RAB27B
with human disease.