N-glycans have been prepared from Pichia yeast, but their specificities and pharmacological activities were not investigated. Current glycoprotein therapeutics are usually produced by particular human or mammalian cells, and their N-glycans vary according to the activities of glycosyltransferases in their ER-Golgi systems. CHO cells can express more extensively sialylated glycoproteins with molecular weights higher than those produced by HEK293, and in some cases may retard the secretion of the recombinants. The present study found that ATB fusion proteins were not secreted from CHO-K1 cells that could be attributed to the unfavorable glycosylation in the cells. We have, for the first time, expressed recombinant venom fibrinogenases with different N-glycans from animal cells, while conserving the protein sequences and the glycosylation sites. HKATB cleaved the Aa, the Bb, and the c chains of human and rabbit fibrinogen, but its cleavage sites on human fibrinogen became less specific and the enzyme did not release FpA. Moreover, HKATB clotted human plasma slower than acutobin did, and was less effective than acutobin in reducing fibrinogen levels in mice. SWATB showed lower stability and lower activities for human fibrinogen. The Nglycans of SWATB possibly are too bulky than is optimal for the enzyme to bind and cleave human fibrinogen. Thus, the fibrinogen specificities and enzymatic activities of ATBs could be affected by the size and branching of their glycan structures, the safety and clinical efficacy of using ATBs in place of native acutobin are questionable at present. The amino acid sequence identities among SVTLEs are about 60% or higher, but their sequence identities to thrombin are less than 30%. Most SVTLEs are fibrinogenases and do not show multiple biological activities as thrombin does, and their clotting efficacies are much lower than thrombin. Moreover, each SVTLE could show rather different chain-selectivities or preferences for fibrinogens from different species. Previous attempts to correlate the amino acid sequences of SVTLEs with their fibrinogen-chain specificities had only limited success. It was postulated that the charge distribution profiles together with the topographic variations around the catalytic interface define the selectivity of SVTLEs for various plasma proteins. The interaction of SVTLEs with macromolecular substrates is certainly not only dependent on their catalytic sites. Notably, most of the SVTLEs are highly glycosylated relative to other venom enzymes. This may imply certain associations between fibrinogen and the glycans of a SVTLE during the venom evolution. Thrombin possesses charged exosites and so possibly also the venom enzymes. The contribution of sugars to the interactions between a glycoprotein enzyme and its substrate could be achieved by specific structural component of the glycans or the sugar clustering effects.