Efficient chemoenzymatic synthesis of sialyl Tn-antigen and derivatives

Abstract

An N-terminal and C-terminal truncated recombinant α2–6-sialyltransferase cloned from Photobacterium sp. JH-ISH-224, Psp2,6ST(15–501)-His₆, was shown to be an efficient catalyst for one-pot three-enzyme synthesis of sialyl Tn (STn) antigens and derivatives containing natural and non-natural sialic acid forms.

Increased sialylation of cell surface glycoconjugates in malignant tumors has been shown to correlate to the invasive and metastatic growth of carcinoma cells.¹ Many sialic acid-containing glycans are considered as tumor-associated carbohydrate antigens (TACAs)² including sialyl Tn, sialyl T, sialyl Lewis^x, and sialyl Lewis^a, etc.³ Among TACAs, sialyl Tn antigens (Siaα2–6GalNAcα1-O-Ser/Thr) are of particular interest.⁴ The level of STn expression is limited on normal cells but is elevated on a wide range of cells of breast, prostate, pancreas, colorectal, lung, gastric, and ovarian cancers.⁵ The expression of STn has also been identified as an independent indicator for poor prognosis of cancer.^4a,4b Therefore, STn is considered a specific tumor-associated antigen for vaccination. The conjugate of STn antigen with the keyhole limpet hemocyanin (KLH) has been developed as a therapeutic vaccine (Theratope®) for treatment of metastatic breast cancer.⁶ It has been shown that STn with modified sialic acid forms such as those with N-iso-butanoyl, N-phenylacetyl,⁷ or fluorine⁸ substituent can significantly increase anti-STn IgG titers and induce strong T-cell-mediated immune response in patients.

STn antigens have been synthesized via various chemical approaches.⁹ These methods require multi-step protection and deprotection processes. In addition, the chemical sialylation step often leads to low yields due to difficulties in controlling the stereospecificity of α-sialyl linkage. On the other hand, chemoenzymatic method using sialyltransferase-catalyzed reaction can provide high regio- and stereo- selectivity. Nevertheless, despite of its good expression level in E. coli (36 mg/L culture) and high efficiency in sialylation of β-linked galactosides, a previously well characterized recombinant Photobacterium damselae α2–6-sialyltransferase (Pd2,6ST)¹⁰ is much less efficient in using α-N-acetylgalactosamine (GalNAc)-linked glycosides as acceptors in synthesizing STn antigen and derivatives.^10a,10c,11 To improve the yields for the synthesis of STn antigens using chemoenzymatic approaches, we aimed to identify an α2–6-sialyltransferase with good solubility, high expression level in E. coli, and has good activity towards α-GalNAc glycosides as acceptors. These criteria are especially important for large scale enzymatic synthesis of STn or preparative synthesis of a library of STn derivatives with diverse sialic acid forms and various aglycons.

Upon analyzing several recently reported bacterial α2–6-sialyltransferases,¹² we identified the α2–6-sialyltransferase from Photobacterium sp. JH-ISH-224 (Psp2,6ST)^12a,12b as our candidate. Psp2,6ST shares 54% amino acid sequence identity with Pd2,6ST and has better expression in E. coli compared to the α2–6-sialyltransferase from Photobacterium leiognathi JT-SHIZ-145.^12c Also, it does not have the sialidase activity reported for the α2–6-sialyltransferase from P. leiognathi JT-SHIZ-119.^12d Using a synthetic gene of Psp2,6ST with codons optimized for E. coli expression system as the template for polymerase chain reaction (PCR), a truncated C-His₆-tagged fusion protein with deletions at both N-terminus (deleting 2–14 amino acid residues) and C-terminus (deleting 502–514 amino acid residues) was cloned into pET22b(+) vector. Expression in E. coli BL21 (DE3) cells at 20 °C for 20 h with vigorous shaking (250 rpm) after induction with 0.3 mM of isopropyl-1-thio-β-D-galactoside (IPTG) followed by Ni²⁺-NTA column purification routinely provided 25 mg of pure, soluble and active Psp2,6ST(15–501)-His₆ per liter of E. coli culture.

The α2–6-sialyltransferase activity of Psp2,6ST(15–501)-His₆ was investigated under different pH conditions using three fluorescent-labeled acceptors including 4-umbelliferyl β-lactoside (LacβMU),¹³ GalNAcα2AA (11), and GalNAcαSer (12). As shown in Figure S3 in the Supporting Information, the optimal pH ranges for Psp2,6ST(15–501)-His₆ were pH 7.5–9.0 and pH 7.5–8.0, respectively, when LacβMU and GalNAcαSer were used as acceptor substrates. The activity declined quickly when pH was below 6.0 or above 9.0. However, when GalNAcα2AA was used as the acceptor, the optimal pH range was pH 5.0–6.0. The enzyme activity decreased quickly below pH 5.0 but only decreased moderately with the increase in the pH range of 6.0–10.0. For comparison, His₆-Pd2,6ST(16–497) was active in a wide pH range of 6.0–9.0 when GalNAcα2AA or GalNAcαSer was used as an acceptor. This was similar to the pH profile of the enzyme when LacβMU was used as an acceptor.^10a Figure S3 also clearly shows that when GalNAcα2AA or GalNAcαSer was used as an acceptor, Psp2,6ST(15–501)-His₆ was a more efficient catalyst than His₆-Pd2,6ST(16–497).

Kinetic studies (Table 1) confirmed that in general, Psp2,6ST(15–501)-His₆ is a more reactive α2–6-sialyltransferase than His₆-Pd2,6ST(16–497). The catalytic efficiency (k_cat/K_m) of Psp2,6ST(15–501)-His₆ was 1.6-, 36.7-, 1.6-, and 3.4-fold higher than that of His₆-Pd2,6ST(16–497) when LacβMU,^10b GalNAcα2AA (11), GalNAcαSer (12), and GalNAcαThr (13) were used as the acceptor substrates respectively. The differences in the catalytic efficiencies were mainly caused by the lower K_m values of different acceptors for Psp2,6ST(15–501)-His₆ compared to His₆-Pd2,6ST(16–497). When GalNAcα2AA (11), GalNAcαSer (12), and GalNAcαThr (13) were used as acceptors, higher k_cat values of Psp2,6ST(15–501)-His₆ also contributed to the better catalytic efficiencies especially for the case of GalNAcα2AA (11) for which a 6-fold better k_cat value was observed. The kinetic studies thus demonstrated that Psp2,6ST(15–501)-His₆ is a promising catalyst for synthesizing sialyl Tn-antigens.

Table 1.

Apparent kinetic parameters of Psp2,6ST(15–501)-His₆ and His₆-Pd2,6ST(16–497).

Enzymes	Psp2,6ST(15–501)-His6			His6-Pd2,6ST(16–497)
Substrates	Km (mM)	kcat (min−1)	kcat/Km (mM−1 min−1)	Km (mM)	kcat (min−1)	kcat/Km (mM−1 min−1)
CMP-Neu5Ac	0.62±0.04	(1.4±0.1)×102	2.2×102	0.90±0.20	(1.6±0.1)×102	1.7×102
LacβMU	0.36±0.07	(1.0±0.1)×102	2.9×102	a0.80±0.10	a(1.4±0.1)×102	a1.8×102
CMP-Neu5Ac	3.9±0.4	4.8±0.2	1.2	11±1	0.38±0.01	0.034
GalNAcα2AA	9.5±2.7	31±5	3.3	51±5	4.7±0.2	0.09
GalNAcαOSer	1.4±0.1	34±3	24	1.6±0.2	24±1	15
GalNAcαOThr	5.9±1.6	16±1	2.7	10±1	8.3±0.2	0.8

^aData were cited from a paper reported previously.^10b

Using Psp2,6ST(15–501)-His₆ with a Pasteurella multocida sialic acid aldolase¹⁴ and an N. meningitidis CMP-sialic acid synthetase (NmCSS)¹⁵ in an efficient one-pot three-enzyme sialylation system established in our lab (Scheme 1),¹⁶ sialyl Tn-antigens and derivatives containing different sialic acid forms and various aglycons were synthesized effectively in preparative scales.

Scheme 1.

Scheme for one-pot three-enzyme chemcoenzymatic synthesis of sialyl Tn antigens containing different sialic acid forms. Enzymes: Pm aldolase, P. multocida sialic acid aldolase; NmCSS, N. meningitidis CMP-sialic acid synthetase; Psp2,6ST, Photobacterium sp. JH-ISH-224 α2–6-sialyltransferase.

To synthesize sialyl Tn analogs containing different sialic acid forms, the reactions were carried out at 37 °C in a Tris-HCl buffer (100 mM) containing Mg²⁺, pyruvate, CTP, a sialic acid precursor, 3-azidopropyl N-acetamido α-galactoside 10 (GalNAcαProN₃) as a sialyltransferase acceptor, and three enzymes. N-Acetylmannosamine 1 (ManNAc), N-glycolylmannosamine 2 (ManNGc), mannose 3, and their chemically synthesized derivatives 4–9 were used as sialic acid precursors. As shown in Table 2, Psp2,6ST(15–501)-His₆ exhibited promiscuous donor substrate specificity and was able to catalyze the transfer of different sialic acids formed from 1–9 to GalNAcαProN₃ 10 to produce STn analogs 14–22 containing Neu5Ac, Neu5Gc, and other sialic acid forms. The sialosides containing natural occurring sialic acid forms, Neu5Acα2–6GalNAcαProN₃ 14, Neu5Gcα2–6GalNAcαProN₃ 15, Kdnα2–6GalNAcαProN₃ 16, Neu5GcMeα2–6GalNAcαProN₃ 17, and Neu5GcAcα2–6GalNAcαProN₃ 18 were obtained in 60% to 86% yields from ManNAc 1, ManNGc 2, mannose 3, ManNGcMe 4, and ManNGcAc 5 as six-carbon sialic acid precursors, respectively. Non-natural STn-antigen analogs Neu5AcN₃α2–6GalNAcαProN₃ 19, Neu5AcFα2–6GalNAcαProN₃ 20, Neu5GcBnα2–6GalNAcαProN₃ 21, and Neu5AcCbzα2–6GalNAcαProN₃ 22, which contain different function groups at the C-5 position of the terminal sialic acid residue, were also obtained in good yields (67–80%) from C2-modified ManNAc derivatives 6–9. The excellent yield of compound 20 (80%) indicated that the fluorine modification on the N-acetyl group of Neu5Ac did not affect the sialyltransferase activity of Psp26ST. Fluorine-modification on STn antigen has been shown to significantly increase anti-STn IgG titers and improve the ratios of anti-STn IgG/IgM.⁸ Therefore, fluorinated compounds which provide useful candidates for vaccine development can be obtained efficiently by chemoenzymatic synthesis without compromising the synthetic yield.

Table 2.

One-pot three-enzyme synthesis of STn antigens and derivates.

Donors	Acceptors	Products	Yields (%)
			76
	10		75
	10		60
	10		86
	10		68
	10		75
	10		80
	10		67
	10		72
1			64
1			72
1			51

STn antigens and analogs containing the most common sialic acid form Neu5Ac and various aglycons were also synthesized. As shown in Table 1, preparative-scale sialylation of GalNAcα2AA 11 by Neu5Ac formed from ManNAc and pyruvate successfully produced the fluorescent-labeled sialylated product Neu5Acα2–6GalNAcα2AA 23 in 64% yield. The one-pot three-enzyme sialylation of biologically important Tn antigens GalNAcαSer 12 and GalNAcαThr 13 was accomplished with 72% and 51% yields, respectively, to produce desired Neu5Acα2–6GalNAcαSer 24 and Neu5Acα2–6GalNAcαThr 25. In comparison, a previous synthesis of a compound similar to 25 from GalNAcαThr using the trans-sialidase activity of V. cholerae and C. perfringens sialidases resulted in low yields (16% and 10% respectively).¹⁷ Compounds 24 and 25 can be used as building blocks in the synthesis of STn glycopeptides. Alternatively, Psp2,6ST(15–501)-His₆ may also be used to improve the synthetic yields of STn peptides via α2–6-sialylation of Tn peptides previously catalyzed by His₆-Pd2,6ST(16–497)^10c.

In summary, we improved the expression of Psp2,6ST in E. coli by using a codon-optimized synthetic gene as a template for PCR and introducing an additional C-terminal 13-amino-acid-truncation. The resulting Psp2,6ST(15–501)-His₆ was shown to be a more reactive α2–6-sialyltransferase than His₆-Pd2,6ST(16–497), especially in the synthesis of STn antigens and their derivatives. Psp2,6ST(15–501)-His₆ exhibited promiscuous donor and acceptor substrate specificities and was used successfully in an efficient one-pot three-enzyme sialylation system for synthesizing STn antigens and derivatives containing diverse natural and non-natural sialic acid forms with various aglycons.