Oftedal Olav T, Nicol Stewart C, Davies Noel W, Sekii Nobuhiro, Taufik Epi, Fukuda Kenji, Saito Tadao, Urashima Tadasu
Smithsonian Environmental Research Center, Smithsonian Institution, Edgewater, MD 21037, USA
School of Biological Sciences.
Glycobiology. 2014 Sep;24(9):826-39. doi: 10.1093/glycob/cwu041. Epub 2014 May 7.
The monotreme pattern of egg-incubation followed by extended lactation represents the ancestral mammalian reproductive condition, suggesting that monotreme milk may include saccharides of an ancestral type. Saccharides were characterized from milk of the Tasmanian echidna Tachyglossus aculeatus setosus. Oligosaccharides in pooled milk from late lactation were purified by gel filtration and high-performance liquid chromatography using a porous graphitized carbon column and characterized by (1)H NMR spectroscopy; oligosaccharides in smaller samples from early and mid-lactation were separated by ultra-performance liquid chromatography and characterized by negative electrospray ionization mass spectrometry (ESI-MS) and tandem collision mass spectroscopy (MS/MS) product ion patterns. Eight saccharides were identified by (1)H NMR: lactose, 2'-fucosyllactose, difucosyllactose (DFL), B-tetrasaccharide, B-pentasaccharide, lacto-N-fucopentaose III (LNFP3), 4-O-acetyl-3'-sialyllactose [Neu4,5Ac(α2-3)Gal(β1-4)Glc] and 4-O-acetyl-3'-sialyl-3-fucosyllactose [Neu4,5Ac(α2-3)Gal(β1-4)[Fuc(α1-3)]Glc]. Six of these (all except DFL and LNFP3) were present in early and mid-lactation per ESI-MS, although some at trace levels. Four additional oligosaccharides examined by ESI-MS and MS/MS are proposed to be 3'-sialyllactose [Neu5Ac(α2-3)Gal(β1-4)Glc], di-O-acetyl-3'-sialyllactose [Neu4,5,UAc3(α2-3)Gal(β1-4)Glc where U = 7, 8 or 9], 4-O-acetyl-3'-sialyllactose sulfate [Neu4,5Ac(α2-3)Gal(β1-4)GlcS, where position of the sulfate (S) is unknown] and an unidentified 800 Da oligosaccharide containing a 4-O-acetyl-3'-sialyllactose core. 4-O-acetyl-3'-sialyllactose was the predominant saccharide at all lactation stages. 4-O-Acetylation is known to protect sialyllactose from bacterial sialidases and may be critical to prevent microbial degradation on the mammary areolae and/or in the hatchling digestive tract so that sialyllactose can be available for enterocyte uptake. The ability to defend against microbial invasion was probably of great functional importance in the early evolution of milk saccharides.
先进行卵孵化再进行长期哺乳的单孔目动物模式代表了哺乳动物的原始生殖状况，这表明单孔目动物的乳汁可能包含原始类型的糖类。对塔斯马尼亚针鼹（Tachyglossus aculeatus setosus）的乳汁中的糖类进行了表征。通过凝胶过滤和使用多孔石墨化碳柱的高效液相色谱法对哺乳后期混合乳汁中的寡糖进行纯化，并通过核磁共振氢谱（¹H NMR）进行表征；通过超高效液相色谱法对哺乳早期和中期较小样本中的寡糖进行分离，并通过负电喷雾电离质谱（ESI-MS）和串联碰撞质谱（MS/MS）产物离子模式进行表征。通过¹H NMR鉴定出了八种糖类：乳糖、2'-岩藻糖基乳糖、二岩藻糖基乳糖（DFL）、β-四糖、β-五糖、乳糖-N-岩藻五糖III（LNFP3）、4-O-乙酰基-3'-唾液酸乳糖[Neu4,5Ac(α2-3)Gal(β1-4)Glc]和4-O-乙酰基-3'-唾液酸-3-岩藻糖基乳糖[Neu4,5Ac(α2-3)Gal(β1-4)[Fuc(α1-3)]Glc]。根据ESI-MS，其中六种（除DFL和LNFP3外）在哺乳早期和中期存在，不过有些处于痕量水平。通过ESI-MS和MS/MS检测的另外四种寡糖被认为是3'-唾液酸乳糖[Neu5Ac(α2-3)Gal(β1-4)Glc]、二-O-乙酰基-3'-唾液酸乳糖[Neu4,5,UAc3(α2-3)Gal(β1-4)Glc，其中U = 7、8或9]、4-O-乙酰基-3'-唾液酸乳糖硫酸盐[Neu4,5Ac(α2-3)Gal(β1-4)GlcS，其中硫酸盐（S）的位置未知]和一种含有4-O-乙酰基-3'-唾液酸乳糖核心的未鉴定的800 Da寡糖。4-O-乙酰基-3'-唾液酸乳糖是所有哺乳阶段的主要糖类。已知4-O-乙酰化可保护唾液酸乳糖免受细菌唾液酸酶的作用，对于防止乳腺乳晕和/或幼体消化道中的微生物降解可能至关重要，这样唾液酸乳糖就可以被肠细胞摄取。在乳汁糖类的早期进化过程中，抵御微生物入侵的能力可能具有重要的功能意义。
