The human CST6 gene is a tiny gene. Together with its basic promoter, it spans about 2,500 bp and is flanked in the 5 upstream region by an inverted, 290-bp Alu-Sx(Sq) repeat.

Like most cystatin genes, the human CST6 gene is organized into three exons separated by two introns. Exon-1 is 294-bp long, contains the 5-untranslated region (5-UTR) and the starting ATG codon of the coding sequence. Exon-2 is 126-bp long. Exon-3 is 188-bp long, contains a TGA stop codon, the 3-UTR as well as a typical AATAAA polyadenylation signal followed by 20 bp. Intron-1 and intron-2 are 541- and 365-bp in length, respectively.

The human CST6 gene is transcribed into a single mRNA species of about 607 nucleotides (nt). There are no alternate transcript species. The transcript is composed of a 5-UTR of 53 nt, a coding sequence of 447 nt, and a 3-UTR of 107 nt. A palyndromic structure located some 360 nt downstream of the AUG initiation codon (or 26 codons upstream of the TGA stop codon) seems to be responsible for some sequence variation in that region. Indeed, several expressed sequence tags (ESTs) differ primarily if not solely in that region of the mRNA sequence.
Transcription from the CST6 gene promoter seems to be both constitutive and regulated. Numerous potential SP1 binding sites (TESS/TransFac database v4.0) in the CST6 promoter may account for a low to moderate basal promoter activity in many tissues. High expression occurs only in a few tissues such as the skin, placenta, ovary, pancreas and the lungs. A quite widespread expression of CST6 is also supported by data extracted from gene expression libraries (GEO, GeneNote, GNF Symatlas, CGAP, EST, SAGE, and UniGene eNortherns). However, there are some conflicting data in the literature suggesting that the CST6 mRNA is expressed in a tissue-specific manner mainly if not exclusively in the skin.
Expression from the human CST6 gene is epigenetically silenced in several tumor types (see below). The 5-end of the CST6 gene including exon-1 has an unusually high (≥ 70%) content in G and C nucleotides. As a matter of fact, a typical CpG island spans across the transcription start site (bp +1) from bp -186 to bp +320 and encompasses all of exon-1. Not surprisingly, treatment of tumor cells by histone deacetylase or DNA methyltransferase inhibitors results in re-expression of CST6 at levels similar to those seen in the normal or benign counterparts.
The unusual GC content (~ 80%) of the 5-UTR of the mRNA suggests that CST6 expression might also be regulated at the translational level by eIF-4E.

No pseudogenes have been identified.

The CST6 gene product, Cst6, is a typical secretory protein. It is synthesized as a preprotein with a patent N-terminal signal sequence. The protein is translocated into the rough endoplasmic reticulum where about 30-50% of the nascent Cst6 polypeptides are N-glycosylated. Upon SDS-PAGE, Cst6 harvested from most cell secretions migrates as two major forms, a 14-kDa unglycosylated and a 17- to 18-kDa glycosylated form.

The three-dimensional organization of Cst6 (assuming it is similar to that of chicken egg white cystatin shown in figure 4) is that of a compact five-pleated beta-sheet that partially wraps around a central alpha-helix. It is not clear what role glycosylation of residue N137 fulfills. Perhaps, N-glycosylation promotes binding of the protein to cells and entry into the endosomal/lysosomal system where Cst6 can interact with target proteases.

Cst6 is a cell-secreted protein. In vitro, the majority (> 95%) of the protein accumulates in the media conditioned by the cells. In cells that overexpress Cst6, prominent labeling of the Golgi apparatus can be seen using indirect immunofluorescence cytochemistry.

In the human skin, where localisation of Cst6 has been most carefully explored, the protein is detected in the stratum granulosum of the epidermis, in the outer root sheet of hair follicles, in the secretory coil epithelium of sweat glands, and in the inner, mature cells of sebaceous glands.

Protease Inhibitor Function: The most widely accepted function of cystatins is that of protease inhibitors. The name cystatin further reminds us that these endogenous protease inhibitors target cysteine proteases. In contrast to metallo- and serine proteases that are mostly secreted proteases, most cysteine proteases are confined within cells where optimal pH and redox conditions favor their enzymatic activity. Thus, the majority of intracellular cysteine proteases are inactivated by oxidizing conditions outside the cells. Nevertheless, it is believed that cystatins inhibit cysteine proteases much faster than do oxidizing conditions and, thereby, prevent excessive tissue damage during the release of lysosomal enzymes.
Among the various types of intracellular cysteine proteases, cystatins seem to target preferentially endosomal/lysosomal cysteine proteases of the papain family, such as cathepsin B, cathepsin K/O2, cathepsin L, cathepsin L2/V and cathepsin S. Some cystatins such as Cst6 are double-headed inhibitors and have a second inhibitory site, i.e., N64 in figures 3 and 4 above. Via this alternate inhibitory site, Cst6 is capable of binding and inhibiting legumain-type cysteine proteases such as AEP/mammalian legumain. Cystatins do not inhibit caspases and calpains seem to be regulated in a different manner. Little is known about the inhibitory potential of cystatins towards other types of intracellular cysteine proteases.
Epithelial barrier function: One important function of Cst6 seems to be in the terminal differentiation of stratified squamous epithelial cells and in the formation of cornified envelops. Indeed, ichq mice with a null mutation in the cst6 gene develop neonatal abnormalities in skin cornification and desquamation that resemble Harlequin ichthyoses in humans. However, no alterations in the CST6 gene were found in the DNA of patients with Harlequin ichthyosis. In mice, the lack of Cst6 function leads to severe dehydration and neonatal lethality. Before serving as a substrate to transglutaminases and being deposited into cornified cell envelops, Cst6 is believed to be important in fine-tuning the enzymatic activities of endosomal/lysosomal cysteine proteases such as cathepsin L, cathepsin L2/V and AEP/mammalian legumain. Deregulated activity of these proteases could lead to abnormal activation of transglutaminases and disorders in cornification.

CST6 Gene orthologs:

Species	UniGene ID	Chromosome	Homology
Human	Hs.139389	11q13.1	100%/149 aa
Pig	Ssc.9061	2p16-17	78%/149 aa
Cow	Bt.5468	29	75%/148 aa
Dog	Cfa.23670	18	71%/149 aa
Rat	Rn.9609	1q43	70%/149 aa
Mouse	Mm.36816	19 A (4.0 cM)	69%/149 aa
Worm	Cel.5518	V	13%/143 aa

In 2004, CST6 was coined as a novel candidate tumor suppressor gene for breast carcinoma. Since then, this gene has been identified as a tumor suppressor gene for other cancers such as cancers of the breast, prostate, brain, lung, cervix and melanocytes. In most tumor tissues, CST6 seems to be epigenetically silenced rather than deleted or mutated. However, in one case (see below) more profound alterations in the human CST6 gene have been observed.
Cervical cancer: One out of 19 primary tumors revealed homozygous deletion of exon-1 sequences. Six other primary tumors exhibited point mutations in the CDS of the CST6 gene. Two of these mutations (M34T and L131F) occurred in proximity to the consensus binding sites for cathepsins (figure 6) and resulted in diminished affinity of the mutant inhibitor for cathepsin L.

No germ-line mutations have been detected.