Cloning and Expression of Bovine Sodium/Glucose Cotransporters

Abstract

The Na+-dependent glucose cotransporters (SGLT) are a family of glucose transporters that mediate an active, sodium-linked transport process against an electrochemical gradient. The SGLT are known to play important roles in absorption of dietary D-glucose and D-galactose from the intestinal lumen and in the reabsorption of D-glucose from the glomerular filtrate in kidney. To study the role and regulation of SGLT in tissues of lactating cows, we cloned and sequenced the full-length cDNA of bovine SGLT1 and SGLT5. Open reading frame analysis predicted that bovine SGLT1 is composed of 664 amino acids with a molecular weight of approximately 73 kDa, and SGLT5 is composed of 597 amino acids with a molecular weight of approximately 65 kDa. The deduced amino acid sequence of bovine SGLT1 is 48% identical and 66% conserved relative to that of bSGLT5. The amino acid sequence of bovine SGLT1 is 97, 88, 87, 86, 85, and 84% identical to sheep, mouse, rat, horse, human, and rabbit SGLT1, respectively. In contrast, the amino acid sequence of bSGLT5 is relatively divergent among species, being 85, 64, and 48% identical to rabbit, human, and rat SGLT5, respectively. Bovine SGLT retain the characteristic structural features of SGLT1 proteins described in other species, including membrane-spanning helices and glucose transporter motifs. The major in vitro transcription and translation product of bovine SGLT1 cDNA migrated at an apparent molecular weight of 52 kDa. In the presence of canine microsomal membranes, the translation product increased to 53 kDa, suggesting glycosylation. The SGLT1 mRNA was most abundant in bovine intestine, at intermediate levels in bovine kidney, and at lower levels in bovine mammary gland, liver, and lung. No SGLT1 mRNA was detected in bovine spleen, skeletal muscle, or testes. Expression of SGLT5 mRNA was found predominantly in bovine kidney, only at very low levels in bovine testes, skeletal muscle, and spleen, and was essentially undetectable in bovine mammary gland, liver, lung, and small intestine. Abundance of SGLT1 mRNA in bovine mammary gland increased more than 4-fold during late pregnancy and early lactation. The sequence and expression data reported in this paper lay the groundwork for future studies aimed at unraveling the functional roles of SGLT in supporting milk production and maintaining glucose homeostasis during lactation.

Functional Properties and Genomics of Glucose Transporters

“…- The members of the SGLT family also share considerable homology among the proteins (21-70% amino acid identity to SGLT1) and contain several conserved sodium:solute symporter family signatures that are characteristics of this family (GenBank accession numbers PS00456, PS00457 and PS50283). The SGLTs are predicted to have 14-15 transmembrane helices [3,16,17]. This second structural model is supported by experimental data of glycosylation studies, antibody tagging, mass spectrometry and other methods [3].…”

“…It is predominantly expressed in the brush border membrane of mature enterocytes in the small intestine and plays a major role in absorption of dietary D-glucose and D-galactose from the intestinal lumen [81,82]. However, SGLT1 expression has been observed in many tissues in which glucose is believed to be taken up by the facilitative process, such as the mammary gland and liver [17]. It, thus, may be multifunctional, such as also functioning as a water cotransporter [83].…”

“…The hydrophilic N-terminus is located on the extracellular surface of the plasma membrane. There are several consensus sites for N-linked glycosylation [16,17], however, glycosylation appears not to be required for SGLT1 activity in contrast to GLUT1 [3].…”

Gene expression of six major milk proteins in primary bovine mammary epithelial cells isolated from milk during the first twenty weeks of lactation

“…In this context, many signalling pathways in the lactating pBMEC are known, i.e. the janus kinase/signal transducer and activator of transcription cascade (Darnell, 1997), the growth hormone effects in the mammary mammalian target of rapamycin signalling pathway (Cui et al, 2003), the interaction of insulin and the major milk protein transcription factor E74-like factor 5 (Menzies et al, 2009), and the amino acids and glucose transporters (Zhao et al, 1996(Zhao et al, , 2005. Future research in that field could provide valuable information on improved lactation performance of dairy cows.…”

Associations among milk production traits and glycosylated haemoglobin in dairy cattle; importance of lactose synthesis potential

“…However; a lactating mammary gland cannot synthesize glucose from other precursors due to the absence of glucose-6-phosphatase (Threadgold and Kuhn 1979) and so is dependent on the blood supply for its glucose requirement. Mammary glucose uptake, which is performed by both active and passive transport processes (Zhao et al 1999(Zhao et al , 2005, is independent of the concentration of glucose and insulin in the arterial blood during lactation. It has also been suggested that the blood glucose concentration affects the rate of synthesis of milk lactose, hence milk volume by osmotic association (Kronfeld 1982).…”