The cloning and characterization of the human transcobalamin II gene

Abstract

Transcobalamin II (TCII) is a plasma protein that binds vitamin B12 (cobalamin; Cbl) and facilitates the cellular uptake of the vitamin by receptor-mediated endocytosis. In genetic disorders that are characterized by congenital deficiency of TCII, intracellular Cbl deficiency occurs, resulting in an early onset of megaloblastic anemia that is sometimes accompanied by a neurologic disorder. To define the genetic basis for TCII deficiency, we have cloned and characterized the human gene that encodes this protein. The gene spans a minimum of 18 kbp and contains nine exons and eight introns, with a polyadenylation signal sequence located 509 bp downstream from the termination codon and a transcription initiation site beginning 158 bp upstream from the ATG translation start site. The 5′ flanking DNA does not have a TATA or CCAAT regulatory element, but a 34-nucleotide stretch beginning just upstream of the CAP site contains four tandemly organized 5′-CCCC-3′ tetramers. This sequence is a motif for a trans-active transcription factor (ETF) that regulates expression of the epidermal growth factor receptor gene (EGFR), which also lacks TATA and CCAAT regulatory elements. A GC-rich sequence that binds the SP1 protein is located 356 nucleotides upstream from the first of the series of CCCC tetramers. Although this GC sequence is at an unusual location with respect to the CAP site, a 507-bp fragment containing this GC box drives the chloramphenicol acetyltransferase (CAT) reporter gene after transient transfection into NIH 3T3 cells. No CAT activity was observed when a 420-bp fragment lacking this GC box but containing the ETF-binding domains was similarly transfected into this cell line. One consensus and two atypical motifs for the c-myc ligand are located downstream and upstream, respectively, of the GC box, and this could explain the elevated plasma TCII observed in some patients with multiple myeloma, as the c-myc product is overexpressed in some myeloma cells. Restriction endonuclease digestion of genomic DNA from eight normal subjects with Taq I, Hinfl, Msp I, and Bgl I identified three patterns of restriction fragment length polymorphism (RFLP). A number of the exon/intron splice junctions of human TCII, TCI, and IF genes are located in homologous regions of these proteins, providing evidence that these genes have evolved by duplication of an ancestral gene. This characterization of the TCII gene and the RFLP should facilitate the identification of the mutation(s) responsible for the genetic abnormalities of TCII expression.

Vitamin B12 Gene Polymorphisms and Chronic Diseases

“…transcobalamin I also known as haptocorrin (TCN1) and with intrinsic factor (IF). Regec et al [29] in a comparative analysis of the placement of introns in the coding regions of human TCN2, TCN1 and IF genes, found substantial homology of the amino acids flanking these insertions and together with previous studies this supports the notion that these genes are derived by duplication of an ancestral gene. The authors suggest that despite this homology, a number of important differences in the TCN2 and TCN1 genes indicated that a major event(s) occurred during this evolutional process that randomly translocated the TCN2 gene to chromosome 22 from chromosome 11, where the human TCN1 and IF genes are located.…”

Steroid‐responsive functional B12 deficiency in association with transcobalamin II polymorphism 776C→G

“…The Lazarus‐like improvement in our patient following the administration of steroids, in the context of her previous B12 loading, may have been due to an effect on blocking antibodies. It may be that the intrinsic factor antibodies in our patient were of particularly high affinity and cross‐reacted with TCII (both these proteins share significant amino acid sequence homology (13)), preventing transport of circulating B12 into cells. The administration of steroids might have abrogated this effect, allowing correction of the intracellular metabolic defect.…”

Evaluation of theMYC_CANFAgene in Chinese Shar Peis with cobalamin deficiency

“…The MYC gene family encodes a group of transcription factors that control cell proliferation and differentiation. 4,5 Identification of at least one binding site for the MYC protein, a product of the MYC gene, on the human transcobalamin II gene, 8 showed a possible role of these two gene products in patients with abnormal transcobalamin II concentration and various malignant disorders. [9][10][11] Nevertheless, the data presented here suggest that cobalamin deficiency in Shar Peis is not related to any mutations of the MYC_CANFA gene.…”

“…MYC gene. 8 This may explain why human patients with abnormal transcobalamin II concentration had a variety of different malignant disorders, but especially multiple myeloma and lymphoproliferative disease. [9][10][11] In this context, human studies have shown that patients with transcobalamin II deficiency have a normal total circulating serum cobalamin concentration.…”